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Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Reviewer and Co-Curator: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

The role of ion channels in Na(+)-K(+)-ATPase: regulation of ion
transport across the plasma membrane has been studied by our Team in 2012 and 2013. This is article TWELVE in a 13 article series listed at the end of this article.

Chiefly, our sources of inspiration were the following:

1.            2013 Nobel work on vesicles and calcium flux at the neuromuscular junction

Machinery Regulating Vesicle Traffic, A Major Transport System in our Cells 

The 2013 Nobel Prize in Physiology or Medicine is awarded to Dr. James E. Rothman, Dr. Randy W. Schekman and Dr. Thomas C. Südhof for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells. This represents a paradigm shift in our understanding of how the eukaryotic cell, with its complex internal compartmentalization, organizes the routing of molecules packaged in vesicles to various intracellular destinations, as well as to the outside of the cell. Specificity in the delivery of molecular cargo is essential for cell function and survival. 

http://www.nobelprize.org/nobel_prizes/medicine/laureates/2013/advanced-medicineprize2013.pdf

Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

2. Perspectives on Nitric Oxide in Disease Mechanisms

available on Kindle Store @ Amazon.com

http://www.amazon.com/dp/B00DINFFYC

https://pharmaceuticalintelligence.com/biomed-e-books/series-a-e-books-on-cardiovascular-diseases/perspectives-on-nitric-oxide-in-disease-mechanisms-v2/

3.            Professor David Lichtstein, Hebrew University of Jerusalem, Dean, School of Medicine

Lichtstein’s main research focus is the regulation of ion transport across the plasma membrane of eukaryotic cells. His work led to the discovery that specific steroids that have crucial roles, as the regulation of cell viability, heart contractility, blood pressure and brain function. His research has implications for the fundamental understanding of body functions, as well as for several pathological states such as heart failure, hypertension and neurological and psychiatric diseases.

Physiologist, Professor Lichtstein, Chair in Heart Studies at The Hebrew University elected Dean of the Faculty of Medicine at The Hebrew University of Jerusalem

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/18/physiologist-professor-lichtstein-chair-in-heart-studies-at-the-hebrew-university-elected-dean-of-the-faculty-of-medicine-at-the-hebrew-university-of-jerusalem/

4.            Professor Roger J. Hajjar, MD at Mount Sinai School of Medicine

Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-for-pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/

5.            Seminal Curations by Dr. Aviva Lev-Ari on Genetics and Genomics of Cardiovascular Diseases with a focus on Conduction and Cardiac Contractility

Aviva Lev-Ari, PhD, RN

Aviva Lev-Ari, PhD, RN

Aviva Lev-Ari, PhD, RN and Larry H. Bernstein, MD, FCAP

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Other related research by the Team of Leaders in Pharmaceutical Business Intelligence published on the Open Access Online Scientific Journal

http://pharmaceuticalintelligence.com

See References to articles at the end of this article on

  • ION CHANNEL and Cardiovascular Diseases

https://pharmaceuticalintelligence.com/?s=Ion+Channel

  • Calcium Role in Cardiovascular Diseases – The Role of Calcium Calmodulin Kinase  (CKCaII) and Ca(2) flux
  • Mitochondria and Oxidative Stress Role in Cardiovascular Diseases

Thus, the following article follows a series of articles on ion-channels and cardiac contractility mentioned, above. The following article is closely related to the work of Prof. Lichtstein.

These investigators studied the possible correlation between

  • Myocardial Ischemia (Coronary Artery Disease (CAD)) aka Ischemic Heart Disease (IHD) and
  • single-nucleotide polymorphisms  (SNPs) genes encoding several regulators involved in Coronary Blood Flow Regulation (CBFR), including
  • ion channels acting in vascular smooth muscle and/or
  • endothelial cells of coronary arteries.

They completely analyzed exon 3 of both KCNJ8 and KCNJ11 genes (Kir6.1 and Kir6.2 subunit, respectively) as well as

  • the whole coding region of KCN5A gene (Kv1.5 channel).
The work suggests certain genetic polymorphisms may represent a non-modifiable protective factor that could be used
  • to identify individuals at relatively low-risk for cardiovascular disease
  • an independent protective role of the
    • rs5215_GG against developing CAD and
    • a trend for rs5219_AA to be associated with protection against coronary microvascular dysfunction

Their findings are a lead into further investigations on ion channels and IHD affecting the microvasculature.

Role of genetic polymorphisms of ion channels in the pathophysiology of coronary microvascular dysfunction and ischemic heart disease

BasicResCardiol(2013)108:387   http//dx.dio.org/10.1007/s00395-013-0387-4

F Fedele1•M Mancone1•WM Chilian2•P Severino2•E Canali•S Logan•ML DeMarchis3•M Volterrani4•R Palmirotta3•F Guadagni3

1Department of Cardiovascular, Respiratory, Nephrology, Anesthesiology and Geriatric Sciences,Sapienza University of Rome, UmbertoI Policlinic, Rome, Italy  e-mail:francesco.fedele@uniroma1.it
2Department of Integrative Medical Sciences, Northeastern Ohio Universities College of Medicine, Rootstown,OH
3Department of Advanced Biotechnologies and Bioimaging, IRCCS San Raffaele Pisana,Rome,It
4Cardiovascular Research Unit, Department of Medical Sciences, Centre for Clinical and Basic Research, Raffaele Pisana, Rome, Italy (CBFR)

BasicResCardiol(2013)108:387   http//dx.dio.org/10.1007/s00395-013-0387-4
This article is published with open access at Springerlink.com

Abstract

Conventionally,ischemic heart disease (IHD) study is equated with large vessel coronary disease (CAD). However, recent evidence has suggested

  • a role of compromised microvascular regulation in the etiology of IHD.

Because regulation of coronary blood flow likely involves

  • activity of specific ion-channels, and
  • key factors involved in endothelium-dependent dilation,

genetic anomalies of ion-channels or specific endothelial-regulators may underlie coronary microvascular disease.

We aimed to evaluate the clinical impact of single-nucleotide polymorphisms in genes encoding for

  • ion-channels expressed in the coronary vasculature and the possible
  • correlation with IHD resulting from microvascular dysfunction.

242 consecutive patients who were candidates for coronary angiography were enrolled. A prospective, observational, single-center study was conducted, 

  • analyzing genetic polymorphisms relative to

(1) NOS3 encoding for endothelial nitric oxide synthase (eNOS);
(2) ATP2A2 encoding for the Ca/H-ATP-ase pump (SERCA);
(3) SCN5A encoding for the voltage-dependent Na channel (Nav1.5);
(4) KCNJ8 and in KCNJ11 encoding for the Kir6.1and Kir6.2 subunits
of genetic K-ATP channels, respectively;and
(5) KCN5A encoding for the voltage-gated K channel (Kv1.5).

No significant associations between clinical IHD manifestations and

  • polymorphisms for SERCA, Kir6.1, and Kv1.5. were observed (p[0.05),

whereas specific polymorphisms detected in eNOS, as well as in Kir6.2 and Nav1.5 were found to be correlated with

  • IHD and microvascular dysfunction.

 Interestingly, genetic polymorphisms of ion-channels  seem to have an important clinical impact

  • influencing the susceptibility for microvascular dysfunction and (IHD,
  • independent of the presence of classic cardiovascular risk factors: atherosclerosis   

http//dx.dio.org/10.1007/s00395-013-0387-4

Keywords: Ion-channels, Genetic polymorphisms, Coronary microcirculation, Endothelium, Atherosclerosis Ischemic heart disease

 Introduction

Historically, in the interrogation of altered vascular function in patientswith ischemic heart disease (IHD), scientists have focused their attention on the correlation between

  • endothelial dysfunction and
  • atherosclerosis [11, 53, 6567].

The endothelium-independent dysfunction in coronary microcirculation and its possible correlations with  

  • atherosclerotic disease and
  • myocardial ischemia has not been extensively investigated.

In normal conditions, coronary blood flow regulation (CBFR) is mediated by several different systems, including

  • endothelial,
  • nervous,
  • neurohumoral,
  • myogenic, and
  • metabolic mechanisms [2, 10, 14, 15, 63, 64, 69].

Physiologic CBFR depends also on several ion channels, such as

  • ATP-sensitive potassium (KATP) channels,
  • voltage-gated potassium (Kv) channels,
  • voltage-gated sodium (Nav) channels, and others.

Ion channels regulate the concentration of calcium in both

  • coronary smooth muscle and endothelial cells, which
  • modulates the degree of contractile tone in vascular muscle and
  • the amount of nitric oxide that is produced by the endothelium

Ion channels play a primary role in the rapid response of both

  • the endothelium and vascular smooth muscle cells of coronary arterioles
  • to the perpetually fluctuating demands of the myocardium for blood flow
    [5, 6, 13, 18, 33, 45, 46, 51, 52, 61, 73, 75].

Despite this knowledge, there still exists an important gap about 

  • the clinical relevance and 
  • causes of microvascular dysfunction in IHD

By altering the overall

  • regulation of blood flow in the coronary system,
  • microvascular dysfunction could alter the normal distribution of shear forces in large coronary arteries

Proximal coronary artery stenosis could

  • contribute to microvascular dysfunction [29, 60]. But
  • ion channels play a critical role in microvascular endothelial
  • and smooth muscle function.

Therefore, we hypothesized  that alterations of coronary ion  channels could be the primary cause in a chain of events leading to

  • microvascular dysfunction and 
  • myocardial ischemia

independent of the presence of atherosclerosis.

Therefore, the objective of our study was to evaluate the possible correlation between

  • IHD and single-nucleotide polymorphisms  (SNPs) for genes encoding several regulators involved in CBFR, including
  • ion channels acting in vascular smooth muscle and/or
  • endothelial cells of coronary arteries.

Discussion

Implications of the present work. This study describes the possible correlation of polymorphisms in genes encoding for CBFR effectors (i.e., ion channels, nitric oxide synthase, and SERCA) with the susceptibility for microcirculation dysfunction and IHD.

Our main findings are as follows: (Group 3 – Normal Patients – anatomically and functionally normal coronary arteries).

  • In Group 3, the genotype distribution of SNP rs5215 (Kir6.2/KCNJ11) moderately deviates from the HW equilibrium (p = 0.05).
  • In Group 1 (CAD), the polymorphism rs6599230 of Nav1.5/SCN5A showed deviation from HW equilibrium (p = 0.017).
  • The genotypic distribution of rs1799983 polymorphism for eNOS/NOS3 is inconsistent with the HW equilibrium in groups 1, 2, and 3 (p = 0.0001, p = 0.0012 and p = 0.0001, respectively).

Haplotype analyses revealed that there is no linkage disequilibrium between polymorphisms of the analyzed genes. There was no significant difference in the prevalence of T2DM (p = 0.185) or dyslipidemia (p = 0.271) between groups, as shown in Table2. In regards to genetic characteristics, no significant differences between the three.

1. A marked HW disequilibrium in the genotypic distribution of rs1799983 polymorphism for eNOS/NOS3 was observed in all three populations. Moreover, this SNP seems to be an independent risk factor for microvascular dysfunction, as evidenced by multivariate analysis;
2. The SNPs rs5215_GG, rs5218_CT, and rs5219_AA for Kir6.2/KCJ11 could reduce susceptibility to IHD, since they were present more frequently in patients with anatomically and functionally normal coronary arteries;
3. In particular, with regard to rs5215 for Kir6.2/KCJ11, we observed a moderate deviation from the HW equilibrium in the genotypic
distribution in the control group. In addition, this genotype appears to be an independent protective factor in the development of IHD, as evidenced by multivariate analysis;
4. Furthermore, the trend observed for the SNP  rs5219_AA of Kir6.2/KCNJ11 may suggest an independent protective factor  in the development of coronary microvascular dysfunction
5. The rs1805124_GG genotype of Nav1.5/SCN5A seems to play a role against CAD;
6. No association seems to exist between the polymorphisms of SERCA/ATP2A2, Kir6.1/KCNJ8, and Kv1.5/KCNA5 and the presence of IHD;
7. All groups are comparable regarding the cardiovascular risk factors of T2DM and dyslipidemia, illustrating a potentially important implication of genetic polymorphisms in the susceptibility to IHD.

It is important to underline that the control group (Group 3) is a high-risk population, because of their cardiovascular risk factors

  • hypertension = 17 %,
  • T2DM = 34.1 %,
  • dyslipidemia = 41.4 %,

with an appropriate indication for coronary angiography, in accordance with current guidelines. Nevertheless, these patients were demonstrated to have both anatomically and functionally normal coronary arteries. Moreover, as shown in Tables 2 and 3, we observed that

  • rs5215_GG, rs5218_CT and rs5219_AA for Kir6.2/KCNJ11 had a higher prevalence in this group,compared to patients with CAD
  • and patients with microvascular dysfunction.

Moreover, as shown in Table 4, the presence of the rs5215_GG polymorphism for the Kir6.2 subunit was

  • inversely correlated with the prevalence of cardiovascular risk factors and CAD,whereas
  • rs5219_AA of the Kir6.2 subunit trended towards an inverse correlation with coronary microvascular dysfunction.

On the other hand, the SNP rs1799983_GT of eNOS was

  • confirmed to be an independent risk factor for microvascular dysfunction.

Our data suggest that the presence of certain genetic polymorphisms may represent a non-modifiable protective factor that could be used

  • to identify individuals at relatively low-risk for cardiovascular disease,
  • regardless of the presence of T2DM and dyslipidemia.

Current Clinical and Research Context

In normal coronary arteries, particularly the coronary microcirculation, there are several different mechanisms of CBFR, including

  • endothelial, neural, myogenic, and metabolic mediators [2, 8, 10, 12, 14, 15, 37, 55, 63, 64, 69].

In particular, endothelium-dependent vasodilation acts mainly via eNOS-derived nitric oxide (NO) in response to acetylcholine and shear stress.

  • NO increases intracellular cyclic guanosine monophosphate. It also causes vasodilation via
  • activation of both K-Ca channels and K-ATP channels.

Recent data suggested a pathophysiologically relevant role for the polymorphisms of eNOS/NOS3 in human coronary vasomotion [40–43]. Our data suggest that rs1799983_GT at exon 7 (Glu298Asp, GAG-GAT) of eNOS/NOS3 represents

  • an independent risk factor for coronary micro-vascular dysfunction, which agrees with a recent meta-analysis reporting an
  • association of this SNP with CAD in Asian populations [74]. In addition,
  • this SNP has been associated with endothelial dysfunction, although the mechanisms are not well defined [30].

Consistently, a recent study performed on 60 Indian patients with documented history of CAD reported a significantly higher frequency of rs1799983 (p.05) compared to control subjects, indicating that

  • variations in NOS3 gene may be useful clinical markers of endothelial dysfunction in CAD [54].
Interestingly, another association between rs1799983_GT and impaired collateral development has been observed in patientswith a

  • high-grade coronary stenosis or occlusion [19].
As is well known, the significance of the mechanisms of CBFR is partly determined by the location within the coronary vasculature. For instance, for vessels with a diameter of < 200 µm—which comprise the coronary microcirculation—metabolic regulation of coronary blood flow is considered the most important mechanism [24, 63]. Importantly, many of these mediators of metabolic regulation act through specific ion channels. In particular, in both coronary artery smooth muscle cells and endothelial cells
  • potassium channels determine the resting membrane potential (Em) and serve as targets of endogenous and therapeutic vasodilators [9, 27].
Several types of K+ channels are expressed in the coronary tree.
  • The K-ATP channels couple cell metabolic demand to conductance, via pore-forming (Kir6.1 and/or Kir6.2) subunits and regulatory
    [sulphonylurea-binding (SUR 1, 2A, or 2B)] subunits.
  • Kir6.x allows for channel inhibition by ATP, while SURx is responsible for channel activation by ADP and Mg2+.
K-ATP channel activation results in an outward flux of potassium and

  • consequent hyperpolarization, resulting in
  • voltage-gated calcium channel closure,
  • decreased Ca2+ influx, and ultimately
  • vasodilation [1, 5, 18, 20, 21, 33, 61, 62, 73, 75].

Our data do not support any significant difference regarding the Kir6.1 subunit of the K-ATP channel. On the other hand, this study suggests

  • an important role of specific SNPs for the Kir6.2 subunit (Tables 2, 3)—i.e., rs5215, rs5219, and rs5218—

in the susceptibility to IHD and microvascular dysfunction. These SNPs are among the most studied K-ATP channel polymorphisms, especially in the context of diabetes mellitus. In fact, in both Caucasian and Asian populations, these three SNPs as well as other genetic polymorphisms for the KCNJ11 gene have been associated with diabetes mellitus [34, 35, 44, 50, 57, 58, 70].

Nevertheless, the precise

  • structure–function impacts of the various amino acid substitutions remain unclear.

The rs5215 and rs5219 polymorphisms, also known as I337V and E23K, respectively, are highly linked with reported

  • concordance rates between 72 and 100 % [22, 23, 56].

The high concordance between rs5219 and rs5215 suggests that these polymorphisms

  • may have originated in a common ancestor, further indicating a
  • possible evolutionary advantage to their maintenance in the general population [49].

In our study, multivariate analysis suggests both an independent protective role of the

  • rs5215_GG against developing CAD and
  • a trend for rs5219_AA to be associated with protection against coronary microvascular dysfunction (Table 4a, b).
  • The variant rs5215_GG is a missense SNP located in the gene KCNJ11 at exon 1009 (ATC-GTC) and results in
    the substitution of isoleucine (I) residue with valine (V) [23].

Future studies are necessary to better understand the influence of this single amino acid variant on the function of the channel.

In humans, vasodilation of the coronary microvasculature in response to hypoxia and K-ATP channel opening
  • are both impaired in diabetes mellitus [39].
It is also described that gain-of-function mutations of the KCNJ11 gene cause neonatal diabetes mellitus, and loss-of-function mutations lead to congenital hyperinsulinism [43]. Our study is not discordant with previous studies about the correlation of SNPs of the Kir6.2 subunit and diabetes mellitus. Rather, our findings show that these SNPs are correlated with anatomically and functionally normal coronary arteries,
  • independent of the presence of either diabetes mellitus or dyslipidemia.
These data suggest the possibility that these particular SNPs may identify individuals with decreased risk for coronary microcirculatory dysfunction and IHD,
  • regardless of the presence of T2DM and/or dyslipidemia.

However, further studies are necessary to confirm these findings. In this context, to better investigate the implications of genetic variation in the K-ATP channel,

  • future studies should include ion channel’s functional modification due to the SNPs and analysis of SUR subunits.

More than 40-kV channel subunits have been identified in the heart, and sections of human coronary smooth muscle cells demonstrate Kv1.5 immunoreactivity [16, 17, 27, 38]. Through constant regulation of smooth muscle tone, Kv channels contribute to the control of coronary microvascular resistance [4, 7]. Pharmacologic molecules that inhibit Kv1.5 channels such as

  • pergolide [25],
  • 4-amino-pyridine [32], and
  • correolide [17]

lead to coronary smooth muscle cell contraction and block the coupling between

  • cardiac metabolic demand and
  • coronary blood flow.

However, no significant differences were identified between the study groups in terms of the particular polymorphisms for Kv1.5 that were analyzed in this study. Expression of

  • the voltage-dependent Na+ channel (Nav) has been demonstrated in coronary microvascular endothelia cells [3, 66].

Our analysis reveals a possible implication of the polymorphism rs1805124_GG for Nav1.5 channel with the presence of anatomically and functionally normal coronary arteries. This SNP leads to a homozygous 1673A-G transition, resulting in a His558-to-Arg (H558R) substitution. It is important to underline that

  • our data are the first to correlate the polymorphism rs1805124_GG with IHD.

Further research is necessary to confirm the observed implication.

Finally, we have analyzed the sarco/endoplasmic reticulum calcium transporting Ca2+-ATPase (SERCA), which is fundamental in the regulation of intracellular Ca2+ concentration [6].

SERCA is an intracellular pump that

  • catalyzes the hydrolysis of ATP coupled with the
  • translocation of calcium from the cytosol into the lumen of the sarcoplasmic reticulum.

Although this pump plays a critical role in regulation of the contraction/relaxation cycle, our analysis did not reveal any apparent association between

  • genetic variants of SERCA and the
  • prevalence of microvascular dysfunction or IHD.

Conclusions

This pilot study is the first to compare the prevalence of SNPs in genes encoding coronary ion channels between patients
  • with CAD or microvascular dysfunction and those with both anatomically and functionally normal coronary arteries.
Taken together, these results suggest the possibility of associations between SNPs and IHD and microvascular dysfunction, although

  • the precise manners by which specific genetic polymorphisms affect ion channel function and expression
have to be clarified by further research involving larger cohorts.

Limitations and future perspectives

Notable limitations of this pilot study are as follows:

1. Due to the lack of pre-existing data, the power calculation was performed in advance on the basis of assumptions of allele frequencies and the population at risk.
2. The sample size for each group is small, mainly due to both the difficulty in enrolling patients with normal coronary arteries and normal microvascular function (group 3) and the elevated costs of the supplies such as Doppler flow wires.
3. There is a lack of ethnic diversity of our cohort.
4. Currently, there is an absence of supportive findings in another independent cohort or population. However, our pilot study included patients within a well-defined, specific population and was aimed to identify the presence of statistical associations between selected genetic polymorphisms and the prevalence of a specific disease.
5. There is a lack of functional characterization of the described genetic polymorphisms.
6. We have not identified any correlation between novel SNPs and IHD. Nevertheless, we completely analyzed exon 3 of both KCNJ8 and KCNJ11 genes (Kir6.1 and Kir6.2 subunit, respectively) as well as the whole coding region of KCN5A gene (Kv1.5 channel).  Moreover, we examined previously described SNPs since there are no data in the literature regarding the possible association of the prevalences of those polymorphisms in the examined population.More extensive studies are necessary to confirm our  findings, possibly with a larger number of patients. Future investigations are also required to confirm the roles of ion  channels in the pathogenesis of coronary microvascular dysfunction and IHD. These studies should involve analysis of both other subunits of the K-ATP channels

  • sulfonylurea receptor, SURx and further coronary ion channels (e.g., calcium-dependent K channels), as well as
  • in vitro evaluation of ion channel activity by patch clamp and analysis of channel expression in the human cardiac tissue.

Moreover, to better address the significance of microvascular dysfunction in IHD, it could be interesting to analyze

  • typical atherosclerosis susceptibility genes (e.g., PPAP2B, ICAM1, et al.).

Methods

In this prospective, observational, single-center study – 242 consecutive patients admitted to our department were enrolled with

  • the indication to undergo coronary angiography .

All patients matched inclusion criteria

  1. age [18];
  2. suspected or documented diagnosis of acute coronary syndrome or stable angina
  3. with indication(s) for coronary angiography, in accordance with current guidelines [36, 68], and
  4. the same ethno-geographic Caucasian origin) and

Exclusion Criteria

  1. previous allergic reaction to iodine contrast,
  2. renal failure,
  3. simultaneous genetic disease,
  4. cardiogenic shock,
  5. non- ischemic cardiomyopathy

All patients signed an informed consent document  –

prior to participation in the study, which included

  • acknowledgement of the testing procedures to be performed
    (i.e., coronary angiography; intracoronary tests; genetic analysis, and processing of personal data).

The study was approved by the Institution’s Ethics Committee.
All clinical and instrumental characteristics were collected in a dedicated  database.

 Study Design

(a)  Standard therapies were administered, according to current guidelines [36, 68].
(b) An echocardiography was performed before and after coronary angiography
(c)  Coronary angiography was performed using radial artery or femoral artery
Judkins approach via sheath insertion.
(d) In patients showing normal epicardial arteries, intracoronary functional tests
were performed through Doppler flow wire to evaluate

  1. both endothelium-dependent microvascular function
    [via intracoronary (IC) infusion of acetylcholine (2.5–10 lg)] and
  2. nonendothelium-dependent microvascular function
    [via IC infusion of adenosine (5 lg)] [31]. 

(e) In all enrolled patients, a peripheral blood sample for genetic analysis was taken. 

On the basis  of the  coronary angiography and the intracoronary functional tests, 

  • the 242 patients were divided into three groups (see also Fig. 1).
  1. Group 1: 155 patients with anatomic coronary alteration
    (comprising patients with acute coronary syndrome and chronic stable angina).

    • microvascular dysfunction defined as coronary flow reserve (CFR) \ 2.5
    • after IC infusion of acetylcholine and adenosine].
  2. Group 2: 46 patients with functional coronary alteration
    [normal coronary arteries as assessed by angiography, and

    • as assessed by angiography and with normal functional tests
      (CFR C 2.5 after intracoronary infusion of acetylcholine and adenosine) (Fig. 1).
  3. Group 3: 41 patients with anatomically and functionally normal coronary arteries


BRC 2013 fedele genetic polymorphisms of ion channels.pdf_page_2

Fig. 1 Study design: 242 consecutive not randomized patients matching inclusion and exclusion criteria were enrolled.
In all patients, coronary angiography was performed, according to current ESC/ACC/AHA guidelines. In patients with
angiographically normal coronary artery, intracoronary functional tests were performed. In 242 patients
(155 with coronary artery disease, 46 patients with micro-vascular dysfunction, endothelium and/or non-endothelium
dependent, and 41 patients with anatomically and functionally normal coronary arteries) genetic analysis was performed.

Genetic Analysis

In conformity with the study protocol, ethylenediaminetetraacetic acid (EDTA) whole blood samples were collected according
to the international guidelines reported in the literature [48]. Samples were transferred to the Interinstitutional Multidisciplinary
BioBank (BioBIM) of IRCCS San Raffaele Pisana (Rome) and stored at -80 C until DNA extraction. Bibliographic research by
PubMed and web tools OMIM (http://www.ncbi.nlm.nih.gov/omim), Entrez SNP (http://www.ncbi.nlm.nih.gov/snp), and
Ensembl (http://www.ensembl.org/index.html) were used to select variants of genes involved in signaling pathways

  • related to ion channels and/or reported to be associated with
  • microvascular dysfunction and/or myocardial ischemia and/or
  • diseases correlated to IHD, such as diabetes mellitus.
Polymorphisms for the following genes were analyzed:
  1. NOS3 (endothelial nitric oxide synthase, eNOS),
  2. ATP2A2 (Ca2+/H+-ATPase pump, SERCA2),
  3. SCN5A (voltage-dependent Na+ channel,
  4. Nav1.5),
  5. KCNJ11 (ATP-sensitive K+ channel, Kir6.2 subunit),
  6. KCNJ8 (ATP-sensitive K+ channel, Kir6.1 subunit) and
  7. KCNA5 (voltage-gated K+ channel, Kv1.5).

In particular, we completely analyzed by direct sequencing

  • exon 3 of KCNJ8 (Kir6.1 subunit), which includes eight SNPs, as well as
  • the whole coding region of KCNA5 (Kv1.5 channel), which includes 32 SNPs and
  • four previously described variants [26, 47, 71, 72].
We also examined
  • the whole coding region of KCNJ11 (Kir6.2 subunit), for which sequence variants are described [26, 28].

All SNPs and sequence variants analyzed—a total of 62 variants of 6 genes—are listed in Table 1.

BRC 2013 fedele genetic polymorphisms of ion channels_page_004
BRC 2013 fedele genetic polymorphisms of ion channels_page_005

DNA was isolated from EDTA anticoagulated whole blood using the MagNA Pure LC instrument and theMagNA Pure LC
total DNA isolation kit I (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Standard
PCR was performed in a GeneAmp PCR System 9700 (Applied Biosystems, CA) using HotStarTaq Master Mix
(HotStarTaq Master Mix Kit, QIAGEN Inc, CA). PCR conditions and primer sequences are listed in Table 1.

In order to exclude preanalytical and analytical errors, all direct sequencing analyses were carried out on both
strands using Big Dye Terminator v3.1 Cycle Sequencing kit
(Applied Biosystems), run on an ABI 3130
Genetic Analyzer (Applied Biosystems), and repeated on PCR products obtained from new nucleic acid extractions.
All data analyses were performed in a blind fashion.

Statistical Analysis

This report, intended as pilot study, is the first to compare

  • the prevalence of SNPs in genes encoding  several effectors (including ion channels)
  • involved in CBFR between these groups of patients.

No definite sample size could be calculated to establish a power analysis. groups of patients. However, assuming

  • a 15 % prevalence of normal  macrovascular and microvascular coronary findings in unselected patients
    undergoing coronary angiography,

we estimated that

  • a sample size of at least 150 patients could enable the computation of two-sided 95 % confidence intervals for
    • such prevalence estimates ranging between -5.0 and + 5.0 %.

The significance of the differences of observed alleles and genotypes between groups, as well as

  • analysis of multiple inheritance models for SNPs were also tested
    (co-dominant, dominant, recessive, over-dominant and log-additive)
  • using a free web-based application (http://213. 151.99.166/index.php?module=Snpstats)
  • designed from a genetic epidemiology  point of view to analyze association studies.

Akaike Information Criterion (AIC) was used to determine the best-fitting inheritance model for analyzed SNPs,

  • with the model with the lowest AIC reflecting the best balance of  goodness-of-fit and parsimony.

Moreover,  the allelic frequencies were estimated by gene counting, and the genotypes were scored. For each gene,

  • the observed numbers of each genotype were compared with those expected for a population in Hardy–Weinberg (HW) equilibrium
  • using a free web-based application  (http://213.151.99.166/index.php?module=Snpstats) [59].

Linkage disequilibrium coefficient (D0) and  haplotype analyses were assessed using  the  Haploview 4.1 program.
Statistical analysis was performed using SPSS software package for Windows v.16.0 (SPSS Inc., Chicago, IL).

All categorical variables are expressed as percentages, and all continuous variables as mean ± standard deviation.
Differences between categorical variables

  • were analyzed by Pearson’s Chi-SQ test.

Given the presence of three groups, differences  between continuous variables, were calculated using
(including the number of SNPs tested),

  • one-way ANOVA; a post-hoc analysis with Bonferroni correction was made for multiple comparisons.

Univariate and multivariate logistic regression analyses

  • the independent impact of genetic polymorphisms on
    • coronary artery disease and microvascular dysfunction,

were performed to assess the independent impact of

  • genetic polymorphisms on coronary artery disease
    and microvascular dysfunction
    ,

while adjusting for other confounding variables.  The following parameters were entered into the model:

  • age,
  • male gender,
  • type 2 diabetes mellitus (T2DM),
  • systemic arterial hypertension,
  • dyslipidemia,
  • smoking status, and
  • family history of myocardial infarction (MI).

Only variables with a p value < 0.10 after univariate analysis were entered

  • into the multivariable model as covariates.

A two-tailed p < 0.05 was considered statistically significant.

Definition of Cardiovascular Risk Factors

Patients were classified as having T2DM if they had

  • fasting levels of glucose of >126 mg/dL in two separate measurements or
  • if they were taking hypoglycemic drugs.

Systemic arterial hypertension was defined as

  • systolic blood pressure  > 140 mmHg / diastolic blood pressure > 90 mmHg
  • in two separate measurements or
  • if the patient was currently taking antihypertensive drugs.

Dyslipidemia was considered to be present if

  • serum cholesterol levels were>220 mg/dL or
  • if the patient was being treated with cholesterol-lowering drugs.

Family history of MI was defined as a first-degree relative with MI before the age of 60 years.

Results

Sixty-two polymorphisms distributed among six genes coding for
  • nitric oxide synthase,
  • the SERCA pump, and
  • ion channels
    • were screened for sequence variations using PCR amplification and
    • direct DNA sequencing analysis

in the population of

  • 155 patients with CAD (group 1),
  • 46 patients with microvascular dysfunction (group 2), and
  • 41 patients with normal coronary arteries and
    • normal endothelium dependent and endothelium-independent vasodilation (group 3).
In Group 3, the genotype distribution of

  • SNP rs5215 (Kir6.2/KCNJ11) moderately deviates from the HW equilibrium (p = 0.05).
In Group 1 (CAD), the polymorphism

  • rs6599230 of Nav1.5/SCN5A showed deviation from HW equilibrium (p = 0.017).
The genotypic distribution of groups in terms of polymorphisms for
  • eNOS/NOS3, SERCA/ATP2A2, Nav1.5/SCN5A, Kir6.1/KCNJ8, or Kv1.5/KCNA5
were noticed. However, significant differences (p.05) for the SNPs
  • rs5215_GG, and
  • rs5219_AA of Kir6.2/KCNJ11 were observed,
as shown in Table 2. 

Table 3 displays 
significant differences between normal subjects (group 3) and
  • patients with either CAD (group 1) or microvascular dysfunction (group 2).

BRC 2013 fedele genetic polymorphisms of ion channels_page_006

When correcting for other covariates as risk factors, the rs5215_GG genotype of Kir6.2/KCNJ11 was found to be 

  • significantly associated with CAD after multivariate analysis (OR = 0.319, p = 0.047, 95 % CI = 0.100–0.991), evidencing
  • a ‘‘protective’’ role of this genotype, as shown in Table 4a.

Similarly, a trend that supports this role of Kir6.2/KCNJ11 was also observed

  • in microvascular dysfunction for rs5219 AA. In contrast,
  • rs1799983_GT for eNOS/NOS3 was identified as an independent risk factor

following multivariate analysis (Table 4b), which agrees with literature findings as described below. 

BRC 2013 fedele genetic polymorphisms of ion channels_page_007

SOURCE for TABLES

BasicResCardiol(2013)108:387   http//dx.dio.org/10.1007/s00395-013-0387-4

Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest.
Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

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4. Berwick ZC, Moberly SP, Kohr MC, Morrical EB, Kurian MM, Dick GM, Tune JD (2012) Contribution of voltage-dependent K+ and Ca2+ channels to coronary pressure-
flow autoregulation. Basic Res Cardiol 107:264. doi:10.1007/s00395-012-0264-6
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6. Brini M, Carafoli E (2009) Calcium pumps in health and disease. Physiol Rev 89:1341–1378. doi:10.1152/physrev.00032.2008
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Basic Res Cardiol (2013) 108:387   http://dx.doi.org/10.1007/s00395-013-0387-4

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Read Full Post »


Gene Study of Blood Pressure Response to Dietary Potassium Intervention: Genetic Epidemiology of Salt Sensitivity

Reporter: Aviva Lev-Ari, PhD, RN

Genome-Wide Linkage and Positional Candidate Gene Study of Blood Pressure Response to Dietary Potassium Intervention

The Genetic Epidemiology Network of Salt Sensitivity Study

Tanika N. Kelly, PhD, James E. Hixson, PhD, Dabeeru C. Rao, PhD, Hao Mei, MD, PhD,Treva K. Rice, PhD, Cashell E. Jaquish, PhD, Lawrence C. Shimmin, PhD, Karen Schwander, MS, Chung-Shuian Chen, MS, Depei Liu, PhD, Jichun Chen, MD,Concetta Bormans, PhD, Pramila Shukla, MS, Naveed Farhana, MS, Colin Stuart, BS,Paul K. Whelton, MD, MSc, Jiang He, MD, PhD and Dongfeng Gu, MD, PhD

Author Affiliations

From the Department of Epidemiology (T.N.K., H.M., C.-S.C., J.H.), Tulane University School of Public Health and Tropical Medicine, and Department of Medicine (J.H.), Tulane University School of Medicine, New Orleans, La; Department of Epidemiology (J.E.H., L.C.S., C.B., P.S., N.F., C.S.), University of Texas School of Public Health, Houston, Tex; Division of Biostatistics (D.C.R., T.K.R., K.S.), Washington University School of Medicine, St Louis, Mo; Division of Prevention and Population Sciences (C.E.J.), National Heart, Lung, Blood Institute, Bethesda, Md; National Laboratory of Medical Molecular Biology (D.L.), Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; Cardiovascular Institute and Fuwai Hospital (J.C., D.G.), Chinese Academy of Medical Sciences and Peking Union Medical College and Chinese National Center for Cardiovascular Disease Control and Research, Beijing, China; and Office of the President (P.K.W.), Loyola University Health System and Medical Center, Maywood, Ill.

Correspondence to Dongfeng Gu, MD, PhD, Division of Population Genetics and Prevention, Cardiovascular Institute and Fuwai Hospital, 167 Beilishi Rd, Beijing 100037, China. E-mail gudongfeng@vip.sina.com

Abstract

Background— Genetic determinants of blood pressure (BP) response to potassium, or potassium sensitivity, are largely unknown. We conducted a genome-wide linkage scan and positional candidate gene analysis to identify genetic determinants of potassium sensitivity.

Conclusions— Genetic regions on chromosomes 3 and 11 may harbor important susceptibility loci for potassium sensitivity. Furthermore, the AGTR1 gene was a significant predictor of BP responses to potassium intake.

SOURCE:

Circulation: Cardiovascular Genetics. 2010; 3: 539-547

Published online before print September 22, 2010,

doi: 10.1161/ CIRCGENETICS.110.940635

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Heroes in Medical Research: Dr. Carmine Paul Bianchi Pharmacologist, Leader, and Mentor

Writer/Curator: Stephen J. Williams, Ph.D.

Past articles in this Heroes in Medical Research series had focused on those seemingly small discoveries, sometimes gained serendipitously and through careful observation and experimentation, which led to some of our most important breakthroughs of our time.  I have tried to make the posts more about the people and less about the discoveries

However, though seminal discoveries are so important to the future of science (and should be celebrated), equally if not MORE IMPORTANT is the MENTORING of future scientists and the PROMOTION of fields of study.  One person who exemplified these values was Dr. Carmine Paul Bianchi, who had recently just passed away this August, and will be sorely missed in the field of pharmacology and toxicology.

For those who were not familiar with Dr. Bianchi I have curated some pertinent information about his work as a scientist, professor and Chairman in pharmacology, and leader and spokesperson for the field of pharmacology.  He was one of the founders of the Mid-Atlantic Pharmacology Society and was an advocate and influential in the careers of many pharmacologists and toxicologists.

Comments from fellow colleagues are very welcome (in comment section at end of post)

The following is separated in 3 sections:

  • An obituary from the Philadelphia Inquirer
  •  A section of the history of the Pharmacology Department at Thomas Jefferson University where Dr. Bianchi was Chairman
  • A few important textbooks and scientific articles he had authored

 

Carmine Paul Bianchi, 86, pharmacology professor

Paul Bianchi

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Carmine Paul Bianchi

By Bonnie L. Cook, Inquirer Staff Writer

Posted: August 20, 2013

Carmine Paul Bianchi, 86, of Boothwyn, a professor of pharmacology in Philadelphia for many years, died Tuesday, Aug. 13, of a digestive ailment at Taylor Hospice House in Ridley Park.

Born in Newark, N.J., and raised in Maplewood, Dr. Bianchi served as an Army surgical technician in Tilton General Hospital at Fort Dix from 1945 to 1947.

He earned a bachelor’s degree in chemistry from Columbia University in 1950, a master’s in physiology and biochemistry from Rutgers University in 1953, and a doctorate in physiology and physical chemistry in 1956 from Rutgers.

In the 1950s, he did research at Rutgers and was a public health fellow and visiting scientist at the National Institutes of Health in Maryland.

From 1961 to 1976, he held a number of jobs in the department of pharmacology in the University of Pennsylvania School of Medicine. That culminated in his being named professor of pharmacology.

Dr. Bianchi left in 1976 for Jefferson Medical College of Thomas Jefferson University, where he became pharmacology professor and chairman of the pharmacology department from 1976 to 1987. In 1987, he stepped down from the chairmanship but remained professor of pharmacology. He retired in 1997 as professor emeritus.

Dr. Bianchi was a member of many professional groups, including the New York Academy of Sciences and the American Association for the Advancement of Science.

He was a leader and author in pharmacology, helping edit an industry journal and making himself available for consultation to medical examiners and experts in toxicology.

He wrote or contributed to three books and 200 scientific papers and lectured widely. He enjoyed mentoring medical and graduate students.

His family called Dr. Bianchi “a true renaissance man” who was as comfortable discussing English, history, and politics as he was the sciences.

 

 

 

The following was taken from a history of  Department of Pharmacology  at Thomas Jefferson University  and can be viewed at: http://jdc.jefferson.edu/cgi/viewcontent.cgi?article=1008&context=wagner2

 

 

Carmine Paul Bianchi, Ph.D;

Third Chairman (1976-1986)

The new Chairman of the Department, effective

July 1, 1976, was Carmine Paul Bianchi, Ph.D.

(Figure 8-3) from the University of Pennsylvania

School of Medicine, where he had been Professor

of Pharmacology since [969 and a member of the

faculty of that Department since 1961.

Dr. Bianchi was born on April 9, [927, in

Newark, New Jersey. After receiving his diploma

at Columbia High School in 1945, he spent two

years in the Army Medical Corps as Technical Sgt.

Fourth Grade. He then attended Columbia

University, where he majored in chemistry and

obtained the B.A. degree in 1950. Like Dr.

Gruber, the first Chairman of the Pharmacology

Department at Jefferson, Bianchi earned his Ph.D.

in physiology. He pursued his graduate studies at

Rutgers University, supplementing his physiology

major with a biochemistry minor for the M.S.

degree in [953 and with a physical chemistry minor

for the Ph.D. degree in 1956. Dr. Bianchi then

spent several years at the National Institutes of

Health-two years as a Public Health Fellow and

one as a Visiting Scientist. Following that he was

Assistant Member of the Institute for Muscle

Disease in New York for one year. In 1961 Dr.

Bianchi became classified professionally as a

pharmacologist by becoming an Associate in the

Department of Pharmacology at the University of

Pennsylvania School of Medicine. There he

advanced to Professorship in 1969 and remained

until he came to Jefferson. The evolution of Dr.

Bianchi’s career from physiology to pharmacology

was the logical result of his investigations of the

effect of various drugs on the metabolism and

distribution of some of the important elements of

the body, notably calcium. His major field of

interest became classified and remained in

electrolyte pharmacology.

Throughout his career Dr. Bianchi has been

very active in the affairs of outside professional

organizations. He is a member of the American

Society for Pharmacology and Experimental

Therapeutics, the American Physiological Society,

the American Chemical Society, and the

International Society of Toxicology, to name

only a few. He served as President of both the

Philadelphia Physiological Society and the John

Morgan Society in the same year (1973-1974), and

of the Philadelphia Chapter of the Society for

Neuroscience (1979-1980). He gave much time

and valuable services as Field Editor for the

Journal of Pharmacology and Experimental

Therapeutics ([970-1979) and as a member of the

Pharmacology Section of the National Board of

Medical Examiners (1981-1985).

After Dr. Bianchi became Chairman no

immediate changes in the general structure and

activities of the Department took place. He

enlarged the Department and filled vacancies

occasioned by the retirement of some faculty

members. The didactic schedules and subject

matter offered to the medical and graduate

students underwent only minor annual changes.

Research activities were augmented by the

addition of Dr. Bianchi’s specialty in electrolyte

pharmacology and the appointments of new staff

members for investigations in that and related

flelds. Through the following decade there was a

marked change in the faculty structure of the

Department. The [975 Jefferson catalogue, for

example, listed 15 faculty appointments in

Pharmacology, of which eight were on a primary

full-time basis with offices and laboratories in the

Department. In 1985 there were 36 faculty

appointments of which eight were on a primary

full-time basis. The large increase in the total

number of faculty resulted from adjunct

appointments from outside organizations and from

secondary appointments of faculty members of the

Clinical Departments at Jefferson. This expansion

reflected a broadening of interests and interactions

on both the scientific and clinical fronts in clinical

pharmacology and clinical toxicology.

A notable addition to the faculty of the

Department in 1978 was Dr. Hyman Menduke

as Professor of Pharmacology

(Biostatistics). After receiving his Ph.D. in

Economic Statistics at the University of

Pennsylvania, Menduke came to Jefferson in 1953

as Assistant Professor of Biostatistics with no

official Departmental affiliation until 1963, when

he was appointed Professor of Preventive

Medicine (Biostatistics). When Dr. Menduke first

came to Jefferson he gave a ten-hour course in

biostatistics to the second-year medical students in

time provided during their pharmacology course.

Through the years his offerings expanded to a

12-hour course for freshman medical students and

introductory and advanced courses for graduate

students. An early and valuable contribution was a

series of individual conferences with graduate

students on the statistical planning of their

research problems and the later analysis of their

data.

 

The interests and activities of the Department in

research in toxicology have been emphasized.

Toxicology continued as an important part of the

research program after Dr. Bianchi became

Chairman in 1976, although under his direction

the major emphasis in research became redirected

toward the general areas of cell pharmacology and

neuropharmacology.

In accord with its continuing research and

teaching activities in toxicology, the Department

starting in 1977 organized a series of annual

workshops on Industrial Toxicology sponsored by

the College of Graduate Studies. These were

four-day symposia on important toxicologic

problems in industry and the general environment,

presented by toxicologically involved Jefferson

faculty and by invited experts from other

universities, industry, and government.

In 1979 the Department was awarded a training

grant in Industrial and Environmental Toxicology

by the National Institute of Environmental Health

Sciences. The purpose of this award was to

provide postdoctoral training in toxicology for

individuals who had previously received their

Ph.D. degrees in other sciences. Ten M.S. degrees

were subsequently awarded in this program

through the years from 1981 to 1986.

On December 14, 1978, a full day’s workshop

with outside invited experts was held to discuss

the formation of a Toxicology Center and the

establishment of a Chair in Toxicology-Pathology

to broaden the base of research and training in

toxicology at Jefferson. It was envisioned that the

Center would be an administrative Division within

the Department of Pharmacology, with research

participation from other basic science departments

and the Department of Medicine. Although funds

accumulated in support of a Toxicology Center,

disagreements developed relating to the

administrative base of the Center.

 

A few articles from Dr. Bianchi showing the diversity of his research interests including calcium mobilization, neurotoxicology, and cellular metabolism and physiology.

Muscle fatigue and the role of transverse tubules.

Bianchi CP, Narayan S.

Science. 1982 Jan 15;215(4530):295-6. No abstract available.

 

Effect of adenosine on oxygen uptake and electrolyte content of frog sartorius muscle.

Prosdocimi M, Bianchi CP.

J Pharmacol Exp Ther. 1981 Jul;218(1):92-6.

 

The effect of diazepam on tension and electrolyte distribution in frog muscle.

Degroof RC, Bianchi CP, Narayan S.

Eur J Pharmacol. 1980 Aug 29;66(2-3):193-9.

 

Steady state maintenance of electrolytes in the spinal cord of the frog.

Bianchi CP, Erulkar SD.

J Neurochem. 1979 Jun;32(6):1671-7. No abstract available.

An in-vitro model of anesthetic hypertonic hyperpyrexia, halothane–caffeine-induced muscle contractures: prevention of contracture by procainamide.

Strobel GE, Bianchi CP.

Anesthesiology. 1971 Nov;35(5):465-73. No abstract available.

 

The effects of psychoactive agents on calcium uptake by preparations of rat brain mitochondria.

Tjioe S, Haugaard N, Bianchi CP.

J Neurochem. 1971 Nov;18(11):2171-8. No abstract available.

 

The effect of veratridine on sodium-sensitive radiocalcium uptake in frog sartorius muscle.

Johnson P, Bianchi CP.

Eur J Pharmacol. 1971 Sep;16(1):90-9. No abstract available.

 

The function of ATP in Ca2+ uptake by rat brain mitochondria.

Tjioe S, Bianchi CP, Haugaard N.

Biochim Biophys Acta. 1970 Sep 1;216(2):270-3. No abstract availabl

 

The effects of pH gradients on the uptake and distribution of C14-procaine and lidocaine in intact and desheathed sciatic nerve trunks.

Strobel GE, Bianchi CP.

J Pharmacol Exp Ther. 1970 Mar;172(1):18-32. No abstract available

 

 

More articles by CP Bianchi  can be found at: http://www.ncbi.nlm.nih.gov/pubmed/?term=Bianchi%20CP[auth]

The following is one of the seminal books Dr. Bianchi authored:

 

Role of Calcium Channels of the Sarcolemma and the Sarcoplasmic Reticulum in Skeletal Muscle Functions

http://link.springer.com/article/10.1007%2F978-1-4615-3362-7_17/lookinside/000.png

AND

Advances in General and Cellular Pharmacology (1976)

Toshio Narahashi; Carmine Paul Bianchi

The author of the Advances in General and Cellular Pharmacology is Toshio Narahashi; Carmine Paul Bianchi – very good writer. You can download this e-book absolutely for free. This ebook’s ISBN number is 9781461582007. if you were searching for for free download of kindle books, google books, free pdf books, pdf ebooks, e-books, pdf files or pdf ebooks just stay here for a while, download what you wanted for free and enjoy!

Advances in General and Cellular Pharmacology – Toshio Narahashi; Carmine Paul Bianchi – PDF Free Download Ebook also for Kindle

 

Other articles in this series published on this site include:

Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer

Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin

Volume Two: Interviews with Scientific Leaders

 

 

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The Role of Tight Junction Proteins in Water and Electrolyte Transport

 

Reviewer and Curator: Larry H. Bernstein, MD, FCAP 

 

This article is Part II of a series that explores the physiology, genomics, and the proteomics of water and electrolytes in human and mammalian function in health and disease.  In this portion of curation, we examine the role of special proteins at the tight junctions of cells, including the claudins.  Consistent with the exploration of cation homeostasis, the last featured article is one of the altered handling of calcium (Ca2+) in CHF, and the closely regulated calcium efflux by the sodium-calcium exchanger (NCX).

The Role of Aquaporin and Tight Junction Proteins in the Regulation of Water Movement in Larval Zebrafish (Danio rerio).

Kwong RW, Kumai Y, Perry SF.
Department of Biology, University of Ottawa, Ottawa, Ontario, Canada.
PLoS One. 2013 Aug 14;8(8):e70764.
http://dx.doi.org/10.1371/journal.pone.0070764   eCollection 2013.

Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP) and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio).

We observed that the half-time for saturation of water influx (K u) was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM). Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H(+)-ATPase-rich cells or Na(+)/K(+)-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation.

Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca(2+)-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP); the paracellular routes may become significant when the paracellular permeability is increased.

PMID:  23967101  PMCID: PMC3743848    http://www.ncbi.nlm.nih.gov/pubmed/23967101

The tight junction protein claudin-b regulates epithelial permeability and sodium handling in larval zebrafish, Danio rerio.

Kwong RW, Perry SF.
Department of Biology, University of Ottawa, Ottawa, Ontario, Canada. wkwong@uottawa.ca
Am J Physiol Regul Integr Comp Physiol. Apr 1, 2013; 304(7):R504-13. http://dx.doi.org/10.1152/ajpregu.00385.2012  Epub 2013 Jan 30.

The functional role of the tight junction protein claudin-b in larval zebrafish (Danio rerio) was investigated. We showed that claudin-b protein is expressed at epithelial cell-cell contacts on the skin. Translational gene knockdown of claudin-b protein expression caused developmental defects, including edema in the pericardial cavity and yolk sac.

Claudin-b morphants exhibited an increase in epithelial permeability to the paracellular marker polyethylene glycol (PEG-4000) and fluorescein isothiocyanate-dextran (FD-4). Accumulation of FD-4 was confined mainly to the yolk sac and pericardial cavity in the claudin-b morphants, suggesting these regions became particularly leaky in the absence of claudin-b expression.

Additionally, Na(+) efflux was substantially increased in the claudin-b morphants, which contributed to a significant reduction in whole-body Na(+) levels. These results indicate that claudin-b normally acts as a paracellular barrier to Na(+). Nevertheless, the elevated loss of Na(+) in the morphants was compensated by an increase in Na(+) uptake.

Notably, we observed that the increased Na(+) uptake in the morphants was attenuated in the presence of the selective Na(+)/Cl(-)-cotransporter (NCC) inhibitor metolazone, or during exposure to Cl(-)-free water. These results suggested that the increased Na(+) uptake in the morphants was, at least in part, mediated by NCC. Furthermore, treatment with an H(+)-ATPase inhibitor bafilomycin A1 was found to reduce Na(+) uptake in the morphants, suggesting that H(+)-ATPase activity was essential to provide a driving force for Na(+) uptake. Overall, the results suggest that claudin-b plays an important role in regulating epithelial permeability and Na(+) handling in zebrafish.
PMID: 23364531   http://www.ncbi.nlm.nih.gov/pubmed/23364531

Evidence for a role of tight junctions in regulating sodium permeability in zebrafish (Danio rerio) acclimated to ion-poor water.

Kwong RW, Kumai Y, Perry SF.
Department of Biology, University of Ottawa, Ottawa, ON, Canada. wkwong@uottawa.ca
J Comp Physiol B. Feb 2013 ;183(2):203-13.
http://dx.doi.org/10.1007/s00360-012-0700-9  Epub 2012 Jul 29.

Freshwater teleosts are challenged by diffusive ion loss across permeable epithelia including gills and skin. Although the mechanisms regulating ion loss are poorly understood, a significant component is thought to involve paracellular efflux through pathways formed via tight junction proteins. The mammalian orthologue (claudin-4) of zebrafish (Danio rerio) tight junction protein, claudin-b, has been proposed to form a cation-selective barrier regulating the paracellular loss of Na(+).

The present study investigated the cellular localization and regulation of claudin-b, as well as its potential contribution to Na(+) homeostasis in adult zebrafish acclimated to ion-poor water. Using a green fluorescent protein-expressing line of transgenic zebrafish, we found that claudin-b was expressed along the lamellar epithelium as well as on the filament in the inter-lamellar regions. Co-localization of claudin-b and Na(+)/K(+)-ATPase was observed, suggesting its interaction with mitochondrion-rich cells. Claudin-b also appeared to be associated with other cell types, including the pavement cells. In the kidney, claudin-b was expressed predominantly in the collecting tubules. In addition,

exposure to ion-poor water caused a significant increase in claudin-b abundance as well as a decrease in Na(+) efflux, suggesting a possible role for claudin-b in regulating paracellular Na(+) loss. Interestingly, the whole-body uptake of a paracellular permeability marker, polyethylene glycol-400, increased significantly after prolonged exposure to ion-poor water, indicating that an increase in epithelial permeability is not necessarily coupled with an increase in passive Na(+) loss. Overall, our study suggests that in ion-poor conditions, claudin-b may contribute to a selective reduction in passive Na(+) loss in zebrafish.
PMID: 22843140   http://www.ncbi.nlm.nih.gov/pubmed/22843140

Claudin-16 and claudin-19 function in the thick ascending limb.

Hou J, Goodenough DA.
Washington University School of Medicine, Div Renal Diseases, St Louis, Missouri
Curr Opin Nephrol Hypertens. Sep 2010; 19(5):483-8. http://dx.doi.org/10.1097/MNH.0b013e32833b7125.

The thick ascending limb (TAL) of the loop of Henle is responsible for reabsorbing 25–40% of filtered Na+, 50–60% of filtered Mg2+ and 30–35% of filtered Ca2+. The dissociation of salt and water reabsorption in the TAL serves both to dilute the urine and to establish the corticomedullary osmolality gradient. Active transcellular salt reabsorption results in a lumen-positive transepithelial voltage that drives passive paracellular reabsorption of divalent cations. Claudins are the key components of the paracellular channel. The paracellular channels in the tight junction have properties of ion selectivity, pH dependence and anomalous mole fraction effects, similar to conventional transmembrane channels. Genetic mutations in claudin-16 and claudin-19 cause an inherited human renal disorder, familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC).

In the TAL of Henle’s loop, the epithelial cells form a water-impermeable barrier, actively transport Na+ and Cl− via the transcellular route, and provide a paracellular pathway for the selective absorption of cations. Na+ K+ and Cl− enter the cell through the Na-K-2Cl cotransporter (NKCC2) in the luminal membrane. Na+ exits the cell through the Na+/K+-ATPase, in exchange for K+ entry. K+ is secreted into the lumen through the renal outer medullary potassium channel. Cl− leaves the cell through the basolateral Cl− channel, made up of two subunits, ClCKb and barttin. The polarized distribution of luminal K+ versus basolateral Cl− conductance generates a spontaneous voltage source (Vsp) of +7−8mV , depending on active transcellular NaCl reabsorption. With continuous NaCl reabsorption along the axis of the TAL segment, the luminal fluid is diluted to 30–60mmol/l  and a large NaCl transepithelial chemical gradient develops at the end of the TAL. Because the paracellular permeability of the TAL is cation-selective (with a PNa/PCl value between 2 and 4), the diffusion voltage (Vdi) is superimposed onto the active transport voltage (Vsp) and becomes the major source of the transepithelial voltage (Vte), which now increases up to +30mV.

Early in-vivo micropuncture studies have shown that approximately 50–60% of the filtered Mg2+ is reabsorbed in the TAL. The flux–voltage relationship indicates that Mg2+ is passively reabsorbed from the lumen to the peritubular space through the paracellular pathway in this segment, driven by a lumen positive Vte.  Vte is made of the sum of Vsp and Vdi. There are two prerequisites required for the paracellular Mg2+ reabsorption in the TAL: the lumen-positive Vte as the driving force and the paracellular permeability for the divalent cation Mg2+.

Claudin-16 and claudin-19 underlie familial hypercalciuric hypomagnesemia with nephrocalcinosis

Claudin-16 and claudin-19 play a major role in the regulation of magnesium reabsorption in the thick ascending limb (TAL). This review describes recent findings of the physiological function of claudin-16 and claudin-19 underlying normal transport function for magnesium reabsorption in the TAL. Mutations in the genes encoding the tight junction proteins claudin-16 and claudin-19 cause the inherited human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis. FHHNC, OMIM #248250, is a rare autosomal recessive tubular disorder. As a consequence of excessive renal Mg2+ and Ca2+ wasting, patients develop the characteristic triad of hypomagnesemia, hypercalciuria and nephrocalcinosis. Recurrent urinary tract infections and polyuria/polydipsia are frequent initial symptoms. Other clinical symptoms include nephrolithiasis, abdominal pain, convulsions, muscular tetany, and failure to thrive. Additional laboratory findings include elevated serum parathyroid hormone levels before the onset of chronic renal failure, incomplete distal tubular acidosis, hypocitraturia, and hyperuricemia. In contrast to hypomagnesemia and secondary hypocalcemia (HSH, OMIM #602014), FHHNC is generally complicated by end-stage renal failure in early childhood or adolescence.

Simon et al. used the positional cloning strategy and identified claudin-16 (formerly known as paracellin-1), which is mutated in patients with FHHNC. Most mutations reported to date in claudin-16 are missense mutations clustering in the first extracellular loop composing the putative ion selectivity filter. Konrad et al. have found mutations in another tight junction gene encoding claudin-19 from new cohorts of FHHNC patients (OMIM #248190). The renal tubular phenotypes are indistinguishable between patients with mutations in claudin-16 and those with mutations in claudin-19. Although claudin-16 and claudin-19 underlie FHHNC and paracellular Mg2+ reabsorption in the TAL, the transient receptor potential channel melastatin 6 (TRPM6) regulates the apical entry of Mg2+ into the distal convoluted tubule epithelia. Mutations in TRPM6 cause the HSH syndrome.

These above data suggested the hypothesis that claudin-16 and/or claudin-19 forms a selective paracellular Mg2+/Ca2+ channel, which was tested in a number of in-vitro studies. Ikari et al. transfected low-resistance Madin-Darby canine kidney (MDCK) cells with claudin-16 and reported that the Ca2+ flux in these cells was increased in the apical to basolateral direction, whereas the Ca2+ flux in the opposite direction remained unchanged. The Mg2+ flux was without any noticeable change. Kausalya et al.  transfected the high-resistance MDCK-C7 cell line and found that claudin-16 only moderately increased Mg2+ permeability without any directional preference. The effects of claudin-16 on Mg2+/Ca2+ permeation appeared so small (<50%) that the Mg2+/Ca2+ channel theory incompletely explains the dramatic effect of mutations in claudin-16 on Mg2+ and Ca2+ homoeostasis in FHHNC patients. However, , Hou et al.  transfected the anion-selective LLC-PK1 cell line with claudin-16 and found a large increase in Na+ permeability (PNa) accompanied by a moderately enhanced Mg2+ permeability (PMg). The permeability of claudin-16 to other alkali metal cations was found to be: K+ > Rb+ > Na+.  Yu et al. emphasized that these residue replacements can influence protein structures that may have impacts on ion permeability independent of amino acid charge.

The cation selectivity of the tight junction is vital for generating the lumen positive transepithelial potential in the TAL, which drives paracellular absorption of magnesium. Claudin-16 and claudin-19 require each other for assembly into tight junctions in the TAL. Heteromeric claudin-16 and claudin-19 interaction forms a cation selective tight junction paracellular channel. Loss of either claudin-16 or claudin-19 in the mouse kidney abolishes the cation selectivity for the TAL paracellular pathway, leading to excessive renal wasting of magnesium.

Claudins interact with each other both intracellularly and intercellularly: they copolymerize linearly within the plasma membrane of the cell, together with the integral protein occludin, to form the classical intramembrane fibrils or strands visible in freeze-fracture replicas. These intramembrane interactions (side-to-side) can involve one claudin protein (homomeric or homopolymeric) or different claudins (heteromeric or heteropolymeric). In the formation of the intercellular junction, claudins may interact head-to-head with claudins in an adjacent cell, generating both homotypic and heterotypic claudin–claudin interactions. Using the split-ubiquitin yeast 2-hybrid assay, Hou et al. found strong claudin-16 and claudin-19 heteromeric interaction. The point mutations in claudin-16 (L145P, L151F, G191R, A209T, and F232C) or claudin-19 (L90P and G123R) that are known to cause human FHHNC disrupted the claudin-16 and claudin-19 heteromeric interaction. In mammalian cells such as the human embryonic kidney 293 cells, claudin-16 can be coimmunoprecipitated with claudin-19. Freeze-fracture replicas revealed the assembly of tight junction strands in L cells coexpressing claudin-16 and claudin-19, supporting their heteromeric interaction.

Coexpression of claudin-16 and claudin-19 in LLC-PK1 cells resulted in a dramatic upregulation of PNa and down-regulation of PCl, generating a highly cation-selective paracellular pathway. Certain FHHNC mutations in claudin-16 (L145P, L151F, G191R, A209T, and F232C) or claudin-19 (L90P and G123R) that disrupted their heteromeric interaction abolished this physiological change. As claudin-16 colocalizes with claudin-19 in the TAL epithelia of the kidney, claudin-16 and claudin-19 association through heteromeric interactions confers cation selectivity to the tight junction in the TAL. Human FHHNC mutations in claudin-16 or claudin-19 that abolish the cation selectivity diminish the lumen-positive Vdi as the driving force for Mg2+ and Ca2+ reabsorption, readily explaining the devastating phenotypes in FHHNC patients.  Hou et al. generated claudin-16 deficient mouse models using lentiviral transgenesis of siRNA to knock down claudin-16 expression by more than 99% in mouse kidneys. Claudin-16 knockdown mice show significantly reduced plasma Mg2+ levels and excessive urinary excretions (approximately four-fold) of Mg2+ and Ca2+. Calcium deposits are observed in the basement membranes of the medullary tubules and the interstitium in the kidney of claudin-16 knockdown mice. These phenotypes of claudin-16 knockdown mice recapitulate the symptoms in human FHHNC patients.

The paracellular reabsorption of Mg2+ and Ca2+ is driven by a lumen-positive Vte made up of two components: Vsp and Vdi. When isolated TAL segments were perfused ex vivo with symmetrical NaCl solutions, there was no difference in Vsp between claudin-16 knockdown and wild-type mice, indicating Vsp was normal in claudin-16 knockdown. Blocking the NKCC2 channel with furosemide (thus dissipating VSP), the cation selectivity (PNa/PCl) was significantly reduced from3.1 ± 0.3 in wild type to 1.5 ± 0.1 in claudin-16 knockdown, resulting in the loss of Vdi. When perfused with a NaCl gradient of 145mmol/l (bath) versus 30mmol/l (lumen), the resulting Vdi was +18mV in wild type, but only +6.6mV in claudin-16 knockdown. Thus, the reduction in Vdi accounted for a substantive loss of the driving force for Mg2+ and Ca2+ reabsorption.

Renal handling of Na+ in claudin-16 knockdown mice is more complex. In the early TAL segment, the transcellular and paracellular pathways form a current loop in which the currents traversing the two pathways are of equal size but opposite direction. Net luminal K+ secretion and basolateral Cl− absorption polarize the TAL epithelium and generate Vsp. As the paracellular pathway is cation selective (PNa/PCl=2–4 , the majority of the current driven by Vsp through the paracellular pathway is carried by Na+ moving from the lumen to the interstitium. Hebert et al. estimated that, for each Na+ absorbed through the trans-cellular pathway, one Na+ is absorbed through the paracellular pathway. With the loss of claudin-16 and the concomitant loss of paracellular cation selectivity, Na+ absorption through the paracellular pathway is reduced.  In the late TAL segment, dilution of NaCl in the luminal space creates an increasing chemical transepithelial gradient; back diffusion of Na+ through the cation-selective tight junction generates a lumen-positive Vdi across the epithelium. The paracellular absorption of Na+ will be diminished when Vdi equals Vsp, and reversed when Vdi exceeds Vsp. As an equilibrium potential, Vdi blocks further Na+ backleak into the lumen. Without claudin-16, Vdi will be markedly reduced well below normal, providing a driving force for substantial Na+ secretion. Indeed, claudin-16 knockdown mice had increased fractional excretion of Na+ (FENa) and developed hypotension and secondary hyperaldosteronism. The observed Na+ and volume loss are consistent with human FHHNC phenotypes. For example, polyuria and polydipsia are the most frequently reported symptoms from FHHNC patients.

Epithelial paracellular channels are increasingly understood to be formed from claudin oligomeric complexes. In the mouse TAL, claudin-16 and claudin-19 cooperate to form cation-selective paracellular channels required for normal levels of magnesium reabsorption. Different subsets of the claudin family of tight junction proteins are found distributed throughout the nephron, and understanding their roles in paracellular ion transport will be fundamental to understanding renal ion homeostasis.

Keywords: claudin; hypomagnesemia; thick ascending limb; tight junction; transepithelial voltage.     PMID: 20616717  PMCID: PMC3378375  http://www.ncbi.nlm.nih.gov/pubmed/20616717

Function and regulation of claudins in the thick ascending limb of Henle.

Günzel D, Yu AS.
Depart Clin Physiol, Charité, Campus Benjamin Franklin, Berlin, Germany.
Pflugers Arch. May 2009; 458(1):77-88.
http://dx.doi.org/10.1007/s00424-008-0589-z  Epub 2008 Sep 16.

The thick ascending limb (TAL) of Henle mediates transcellular reabsorption of NaCl while generating a lumen-positive voltage that drives passive paracellular reabsorption of divalent cations. Disturbance of paracellular reabsorption leads to Ca(2+) and Mg(2+) wasting in patients with the rare inherited disorder of familial hypercalciuric hypomagnesemia with nephrocalcinosis (FHHNC). Recent work has shown that the claudin family of tight junction proteins form paracellular pores and determine the ion selectivity of paracellular permeability. Importantly, FHHNC has been found to be caused by mutations in two of these genes, claudin-16 and claudin-19, and mice with knockdown of claudin-16 reproduce many of the features of FHHNC. Here, we review the physiology of TAL ion transport, present the current view of the role and mechanism of claudins in determining paracellular permeability, and discuss the possible pathogenic mechanisms responsible for FHHNC.

Tight junctions form the paracellular barrier in epithelia. Claudins are ~22 kDa proteins that were first identified by Mikio Furuse in the laboratory of the late Shoichiro Tsukita as proteins that copurified in a tight junction fraction from the chicken liver [23]. The observation that they were transmembrane proteins with 4 predicted membrane domains and 2 extracellular domains raised early on the possibility that they could play a key role in intercellular adhesion and formation of the paracellular barrier. In 1999, Richard Lifton’s group identified mutations in a novel gene, which they called paracellin, as the cause of familial hypercalciuric hypomagnesemia, an inherited disorder believed to be due to failure of paracellular reabsorption of divalent cations in the thick ascending limb of the renal tubule. Paracellin turned out to be a claudin family member (claudin-16). This suggested that claudins in general might be directly involved in regulating paracellular transport in all epithelia. This is now supported by numerous studies demonstrating that overexpressing or ablating expression of various claudin isoforms in cultured cell lines or in mice affects both the degree of paracellular permeability and its selectivity (vide infra). Furthermore, in mammals alone there are ~24 claudin genes and each exhibits a distinct tissue-specific, pattern of expression. Thus, the specific claudin isoform(s) expressed in each tissue might explain its paracellular permeability properties.

Each nephron segment expresses a unique set of multiple claudin isoforms, and each isoform is expressed in multiple segments, thus making a complicated picture which even varies between different species. The role of combinations of claudins in determining paracellular permeability properties has hardly been studied yet. In mouse, rabbit and cattle, the thick ascending limb of Henle’s loop is thought to express claudins 3, 10, 11, 16  in adulthood, and, at least in mouse, additionally claudin-6 during development. In addition, claudin-4 has been found in cattle and claudin-8 in rabbit. To date, the distribution of Claudin-19 has been investigated in mouse, rat, and man where its presence in the TAL was demonstrated.

The thick ascending limb (TAL) of Henle’s loop, working as “diluting segment” of the nephron, is characterized by two major properties: high transepithelial, resorptive transport of electrolytes and low permeability to water. Major players to achieve electrolyte transport are the apical Na+-K+-2Cl−symporter (NKCC2), the apical K+ channel ROMK, the basolateral Cl− channel (CLC-Kb) together with its subunit barttin and the basolateral Na+/K+-ATPase . The combined actions of these transport systems have been extensively reviewed and are therefore only briefly summarized here. Na+ and Cl− are resorbed by entering the cells apically through NKCC2 and leaving the cells basolaterally through the Na+/K+-ATPase and CLC-Kb, respectively. In contrast, K+ is either recycled across the apical membrane as it is entering through NKCC2 and leaving through ROMK, or even secreted, as it is also entering the cells basolaterally through the Na+/K+-ATPase. Taking these ion movements together, there is a net movement of positive charge from the basolateral to the apical side of the epithelium, giving rise to a lumen positive voltage (3 – 9 mV [11]; about 5 – 7 mV [30,31]; 7 – 8 mV [57]). Over the length of the TAL, luminal NaCl concentration decreases gradually to concentrations of 30 – 60 mM at the transition to the distal tubule, depending on the flow rate within the tubule (low flow rates resulting in low concentrations).

To keep up such a high gradient, the TAL epithelium has to be tight to water and various studies summarized by Burg and Good report water permeability values from 28 µm/s down to values indistinguishable from zero. Tight junctions of the TAL are, however, highly permeable to cations with PNa being about 2 – 2.7 fold, 2.5 fold or even up to 6 fold that of PCl. Amongst the monovalent cations, a permeability sequence of PK > PNa > PRb = PLi > PCs > Porganic cation was observed which is similar to Eisenman sequence VIII or IX, indicating a strong interaction between the permeating ion and the paracellular pore that enables at least partial removal of the hydration shell (see below). As reviewed by Burg and Good, the transepithelial sodium and chloride permeabilities, estimated from radioisotope fluxes, are high (in the range of 10 – 63·10−6 cm/s) and the transepithelial electrical resistance is correspondingly low (21 – 25 W cm2 ; 30 – 40 W cm2 ; 11 – 34 W cm2 . Blocking active transport by the application of furosemide or ouabain increases transepithelial resistance only slightly, indicating that the low values are primarily due to a very high paracellular permeability. Due to these properties of TAL epithelial cells, Na+ ions leak back into the lumen of the tubule, creating a diffusion (dilution) potential that adds another 10 – 15 mV to the lumen positive potential, so that considerable potential differences (25 mV ; 30 mV; cTAL 23 mV, mTAL 17 mV ) may be reached at very slow flow rates.

Considerable proportions of the initially filtrated Mg2+ (50 – 60%; 50 – 70%; 65 – 75%) and Ca2+ (20%; 30 – 35% are resorbed in the TAL. The transepithelial potential is considered to provide the driving force for the predominantly paracellular resorption of Mg2+ and Ca2+ as in many studies, transport of both divalent ions in the TAL has been found to be strictly voltage dependent (resorbtive at lumen positive potentials, zero at 0 mV and secretory at lumen negative potentials) and permeability considerable (PCa 7.7·10−6 cm/s, i.e. approximately 25% of PNa). There is however, some conflicting evidence, e.g. by Suki et al. and Friedman. Both studies used cTAL (cortical TAL) and found that decreasing the transepithelial potential by applying furosemide did either not alter the unidirectional lumen to bath Ca2+ flux (rabbit) or left a substantial net Ca2+ resorption (mouse). Similarly, Rocha et al. found that bath application of ouabain almost abolished the transepithelial potential, but hardly affected net Ca2+ resorption and conclude that (a) all segments of Henle’s loop are relatively impermeable to calcium and (b) net calcium resorption occurs in the thick ascending limb which cannot be explained by passive mechanisms.  Mandon et al. conclude that both Mg2+ and Ca2+ are transported in the cTAL but not in the mTAL (medullary TAL) of rat and mouse, although transepithelial potential differences were similar in both segments, and even if the transepithelial potential was experimentally elevated to values above 20 mV. Wittner et al. even found evidence that in mouse mTAL the passive permeability to divalent cations is very low and that Ca2+ and Mg2+ can be secreted into the luminal fluid under conditions which elicit large lumen-positive transepithelial potential differences. They conclude that this Ca2+ and Mg2+ transport is most probably of cellular origin. In contrast, in rabbit, both ions are transported along the whole length of the TAL.

Both, Mg2+ and Ca2+ resorption are modulated through the action of the basolateral Ca (and Mg) sensing receptor (CaSR) which is found along the entire nephron but especially in the loop of Henle, distal convoluted tubule (DCT) and the inner medullary collecting duct. Different modes of action on Ca2+ and Mg2+ homeostasis exist, such as an indirect action through the modulation of PTH secretion or direct effects on the cells expressing CaSR. In the TAL the latter model is based on the assumptions depicted above, i.e. that Mg2+ and Ca2+ are resorbed paracellularly, driven by the lumen positive potential, so that a reduction in NaCl resorption causes a reduction in driving force for Mg2+ and Ca2+ resorption. As reviewed by Hebert and Ward, CaSR is activated through an increase in basolateral Ca2+ and/or Mg2+ concentration which triggers an increase in the intracellular Ca2+ concentration. This reduces the activity of the adenylate cyclase which, in turn, inhibits transcellular transport of Na+ and Cl−. In addition, the increase in intracellular Ca2+ activates phospholipase A2 (PLA2) and thus increases the intracellular concentration of arachidonic acid and its derivative, 20-HETE. 20-HETE inhibits NKCC2, ROMK and the Na+/K+-ATPase and by this Mg2+ and Ca2+ resorption. In keeping with this hypothesis, mutations in CaSR affect Ca/Mg resorption. Inactivating mutations cause hypercalcemia, hypocalciuria, hypomagnesiuria and, in some patients hypermagnesemia. Conversely, activating mutations (gain of function mutations) lead to hypocalcemia, hypercalciuria, hypermagnesiuria and in up to 50% of the patients mild hypomagnesemia.

Bartter syndrome type I (mutations in NKCC2), and type II (mutations in ROMK) lead to hypercalciuria and thus cause nephrocalcinosis, but no hypomagnesemia is observed. Reports on hypermagnesiuria are conflicting: while Kleta and Bockenhauer link it to nephrocalcinosis seen in these patients, Rodriguez-Soriano states that patients with neonatal Bartter syndrome (i.e. type I or II) show a lack of hypermagnesiuria that may be explained by compensation in the DCT. Hypomagnesemia is occationally present in Bartter type III (CLC-Kb). However, here it is believed to be mainly due to effects on DCT, where CLC-Kb shows highest expression. Patients with Bartter syndrome IV (CLC-Kbsubunit barttin) may or may not present nephrocalcinosis, while Mg2+ homeostasis appears undisturbed. Interestingly, the largest effects on Mg2+-homeostasis are observed in Gitelman syndrome, a defect in the Na+/Cl− symport (NCC) predominantly found in the DCT, where Mg2+ is transported along the transcellular route. Affected patients suffer from hypomagnesemia, hypermagnesuria and hypocalciuria. The effect on Mg2+ in Gitelman syndrome is still poorly understood and possibly due to a concomitant down-regulation of TRPM6, the apical Mg2+ uptake channel in DCT.

More than 30 different claudin-16 mutations have now been reported in families with FHHNC. Because of the large number of unique mutations, it has not been possible to identify any clear qualitative correlation between the phenotype and individual mutations, although certain mutations are associated with greater severity of disease. In 2006, a second locus was identified, CLDN19, which encodes claudin-19. In the initial report, the phenotype appeared similar to that due to claudin-16 mutations, with the exception that there was a high prevalence of ocular abnormalities, including macular colobomata, nystagmus and myopia. Claudin-19 is normally expressed at high levels in the retina, but why it causes these ocular disorders is unknown.

In vitro studies of claudin function comprise inducible or non-inducible transfection of various cells lines with cDNA for claudins that are not endogenously expressed by the cell line used. Alternatively, cells can be transfected with siRNA directed against an endogenous claudin. In both cases, cells are then grown to confluence on permeable filter supports that allow measurement of transepithelial permeabilities. Before the results of permeability studies can be interpreted, however, several parameters have to be controlled.

First, special care has to be taken to make sure that the exogenous claudin is correctly inserted into the tight junction. This can be achieved e.g. by confocal laser scanning microscopy, colocalizing the claudin of interest  with a tight junction marker protein such as occludin.

Second, it has to be ensured that endogenous claudin expression remains unaffected, as permeability changes always result from the combined effects of alterations in endogenous and exogenous claudins.

Third, it has to be kept in mind that, typically, epithelia express several different claudins that act together to produce tissue specific permeability properties. Thus, ideally, a cell line should be chosen that provides a claudin background resembling that usually experienced by the claudin investigated. The latter two points may be the reason for contradicting results obtained in permeability studies expressing a specific claudin in different cell lines.

Studies of paracellular permeabilities can be divided into two groups, those employing electrophysiological measurements (e.g. determination of diffusion potentials), and those measuring ion or solute flux, using either radioactive isotopes or various analytical methods to determine the amount transported.

Although transepithelial conductances depend on paracellular permeabilities of the predominant ions in the bath solution, conductance changes alone cannot be used to predict ion permeabilities.

Firstly, conductances always depend on both ion and counter-ion, not on one ion species alone.

Secondly, transepithelial conductances are the sum of the conductances of the transcellular and paracellular pathways.

Thus, they only reflect paracellular permeability, if paracellular conductance dominates transepithelial conductance and if transcellular conductance remains constant throughout the experiment. This, however, is often not the case, as concentration changes of the ions investigated may affect transcellular conductance, e.g. by activating ion transporters or by inhibiting ion channels. Thirdly, the specific conductance of each solution employed may differ and has therefore to be assessed and taken into account. Thus, comparison of results from diffusion potential measurements or flux studies and conductance measurements may even yield contradicting results. For the same reasons, other methods based on pure conductance/resistance measurements, including the more sophisticated conductance scanning method or one-path impedance spectroscopy are not ion specific and do not allow the measurement of paracellular permeabilities to single ions.

In contrast to electrophysiological measurements, flux measurements are not limited to ions but can also be extended to uncharged molecules.  Flux measurements per se do not distinguish between transcellular or paracellular transport. Therefore, to estimate paracellular permeabilities, inhibition or at least estimation of the transcellular flux is necessary. Assuming that transcellular flux for energetic reasons is not easily reversible while paracellular flux is passive and thus generally assumed to be symmetric, the transcellular proportion is often estimated by calculating the difference between apical to basolateral and basolateral to apical fluxes. All flux measurements are very sensitive to the development of diffusion zones (“unstirred layers”) near the cells. These layers are depleted/enriched in the compound transported and thus alter the driving forces acting on these compounds, if bath solutions are not continually circulated. If ionic fluxes are investigated, transepithelial potentials may develop that diminish or completely inhibit the flux investigated.

All the techniques described above have been employed to investigate the function of claudin-16 and -19, especially with respect to their ability to increase paracellular permeability to divalent cations. The hypothesis, that claudin-16 (then called paracellin-1) may be a paracellular Mg2+ and Ca2+ pore was originally expressed by Simon et al. It was based on the findings that mutations in claudin-16 were the cause of the severe disturbance in Mg2+ and Ca2+ homeostasis in FHHNC patients together with the observations that claudin-16 is a tight junction protein located in the TAL, i.e. the nephron segment responsible for bulk Mg2+ resoption along the paracellular pathway. When, recently, it was found that claudin-19 mutations were underlying hitherto unexplained cases of FHHNC and that claudin-19 co-localized with claudin-16, the hypothesis was extended to claudin-19.

  1. Cole DEC, Quamme GA. Inherited disorders of renal magnesium handling. J Am Soc Nephrol 2000;11:1937–1947. [PubMed: 11004227]
  2. de Rouffignac C, Quamme G. Renal magnesium handling and its hormonal control. Physiol Rev 1994;74:305–322. [PubMed: 8171116]  
  3. Meij IC, van den Heuvel LP, Knoers NV. Genetic disorders of magnesium homeostasis. BioMetals 2002;15:297–307. [PubMed: 12206395]
  4. Satoh J, Romero MF. Mg2+ transport in the kidney. BioMetals 2002;15:285–295. [PubMed: 12206394]  
  5. Schlingmann KP, Konrad M, Seyberth HW. Genetics of hereditary disorders of magnesium homeostasis. Pediatr Nephrol 2004;19:13–25. [PubMed: 14634861]  
  6. Simon DB, Lu Y, Choate KA, et al. Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption. Science 1999;285:103–106. [PubMed: 10390358]

PMID: 18795318  PMCID:  PMC2666100  http://www.ncbi.nlm.nih.gov/pubmed/18795318

Deletion of claudin-10 (Cldn10) in the thick ascending limb impairs paracellular sodium permeability and leads to hypermagnesemia and nephrocalcinosis.

Breiderhoff T, Himmerkus N, Stuiver M, Mutig K, Will C, Meij IC et al.
Max Delbrück Center for Molec Med, Berlin, Germany. t.breiderhoff@mdc-berlin.de

Erratum in Proc Natl Acad Sci. 2012 Sep 11;109(37):15072.
Proc Natl Acad Sci. Aug 28, 2012; 109(35):14241-6.   http://dx.doi.org/10.1073/pnas.1203834109. Epub 2012 Aug 13.

In the kidney, tight junction proteins contribute to segment specific selectivity and permeability of paracellular ion transport. In the thick ascending limb (TAL) of Henle’s loop, chloride is reabsorbed transcellularly, whereas sodium reabsorption takes transcellular and paracellular routes. TAL salt transport maintains the concentrating ability of the kidney and generates a transepithelial voltage that drives the reabsorption of calcium and magnesium. Thus, functionality of TAL ion transport depends strongly on the properties of the paracellular pathway. To elucidate the role of the tight junction protein claudin-10 in TAL function, we generated mice with a deletion of Cldn10 in this segment. We show that claudin-10 determines paracellular sodium permeability, and that its loss leads to hypermagnesemia and nephrocalcinosis. In isolated perfused TAL tubules of claudin-10-deficient mice, paracellular permeability of sodium is decreased, and the relative permeability of calcium and magnesium is increased. Moreover, furosemide-inhibitable transepithelial voltage is increased, leading to a shift from paracellular sodium transport to paracellular hyperabsorption of calcium and magnesium. These data identify claudin-10 as a key factor in control of cation selectivity and transport in the TAL, and deficiency in this pathway as a cause of nephrocalcinosis.

Whereas regulation of transporters and channels involved in trans-cellular ion transport has been characterized in much detail, the functional and molecular determinants of paracellular ion trans­port in the kidney remain incompletely understood. In the thick ascending limb (TAL) of Henle’s loop, both trans-cellular and paracellular ion transport pathways contribute to reabsorption of Na+, Cl, Mg2+, and Ca2+. Na+ and Clare reabsorbed mostly transcellularly by the concerted action of chan­nels and transporters. Mutations in five of the genes involved lead to Bartter syndrome, a disorder characterized by salt wasting and polyuria. Whereas Clis transported exclusively transcellularly, 50% of the Na+ load, as well as Ca2+ and Mg2+, are reabsorbed via paracellular pathways. In the TAL, this paracellular route is highly cation-selective. The paracellular passage is largely controlled by the tight junction (TJ), a supramolecular structure of membrane-spanning proteins, their intracellular adapters, and scaffolding proteins. Claudins, a family comprising 27 members, are the main components of the TJ defining the permeability properties. They interact via their extracellular loops with corre­sponding claudins of the neighboring cell to allow or restrict pas­sage of specific solutes (5, 6). In the kidney, their expression pattern is closely related to the corresponding segment-specific solute reabsorption profile. Several claudins are expressed in the TAL, including claudin-16, -19, -10, -3, and -18 The importance of claudin-16 and -19 in this tissue is documented by mutations in CLDN16 and CLDN19, which cause familial hypomagnesemia, hypercalciuria, and nephrocalcinosis, an autosomal recessive dis­order that leads to end-stage renal disease. The relevance of CLDN16 for paracellular reabsorption of Mg2+ and Ca2+ was confirmed in mouse models with targeted gene disruption. In addition, claudin-14, expressed in the TAL of mice on a high-calcium diet, was identified as negative regulator of claudin-16 function (15), and sequence variants in CLDN14 have been asso­ciated with human kidney stone disease. The functional significance of claudin-10, which is also ex­pressed in the TAL, remains unclear. This TJ protein is expressed in two isoforms, claudin-10a and claudin-10b, which differ in their first extracellular loop. In cultured epithelial cells, heter-ologous expression of claudin-10a increases paracellular anion transport, whereas claudin-10b expression increases paracellular cation transport. Both isoforms are expressed differentially along the nephron, with claudin-10a found predominantly in cortical segments, whereas claudin-10b is enriched in the medullary region.  In the present study we generated a mouse model with a TAL-specific Cldn10 gene defect to query the role of this protein in renal paracellular in transport in vivo. We found that claudin-10 is crucial to paracellular Na+ handling in the TAL, and that its absence leads to a shift from paracellular sodium transport to paracellular hyperreabsorption of Ca2+ and Mg2+.

Analysis of claudin-10 expression in the kidney. (A) Western blot analysis of kidney membrane extracts from control (ctr) and cKO mice. A dramatic reduction in claudin-10 protein can be seen in kidneys of cKO mice. Levels of the TJ marker occludin are unchanged. (B) Gene expression analysis of Cldn10 variants on cDNA from isolated segments of the nephron. (C) Immunohistological detection of claudin-10 and markers for PCT (NHE3) and TAL (NKCC2) on sections from control mice (ctr) and cKO mice demonstrates no difference in the signal for claudin-10 in the PCT between WT and cKO. Claudin-10 is expressed in TAL tubules positive for NKCC2. No specific clau-din-10 staining is evident in the TAL of cKO mice. Claudin-10 is detected in TJs positive for ZO-1. This signal is absent in cKO mice, whereas ZO-1 staining is unchanged. (Scale bar: 25 μm.)

In control animals, claudin-10 is located mainly in the TAL, as documented by coimmunostaining with the Na+K+2Clcotrans-porter (NKCC2). In this segment, a large portion of the claudin-10 immunofluorescence signal is located outside of the TJ; however, claudin-10 is present in the TJ, as demonstrated by colocalization with the TJ protein ZO-1. PCTs positive for the sodium-proton exchanger NHE-3 showed a considerably weaker signal restricted to the TJ area. Claudin-10 immunoreactivity was virtually absent in NKCC2-positive tubules of cKO mice, in line with the activity of Cre recombinase in this cell type. The immu-noreactivity of claudin-10 in PCTs of cKOs remained unchanged, however. ZO-1 staining in TAL sections of cKOs was unchanged compared with controls, indicating no unspecific effect on TJ structures. The TJ localization of claudin-16 and claudin-19 in medullary rays was similar in cKOs and controls.   To investigate the phenotypic consequences of renal claudin-10 deficiency, we per­formed a histological examination of the kidneys of 10-wk-old cKO mice and their respective controls. Kidneys from cKO mice contained extensive medullary calcium deposits, as revealed by von Kossa and alizarin red S staining. The deposits were found along the outer stripe of the outer medulla. The detection of extensive calcification suggests alterations in renal ion homeostasis in mice deficient for claudin-10.  Serum Na+ and Cllevels and their renal FE excretion rates were not different be­tween genotypes. In addition, serum creatinine and glomerular filtration rate were not altered compared with controls. Taken together, these findings indicate that calcium deposition does not nonpecifically affect overall glomerular or tubular function.

Fig 4. Gene expression analysis of renal claudins (A) and representative renal ion transporters and channels (B) by real-time PCR. Cldn10 deficiency results in differential gene expression of several genes. Values from cKO animals are shown relative to control mice (mean ± SEM). Wnk1, Wnk1-KS, Kcnj1, and Trpm6, n = 5/4; all other genes, n = 10/10. *P < 0.05; **P < 0.01; ***P < 0.001.    The thiazide-sensitive NaCl cotransporter NCC (Slc12a3), the protein involved in NaCl absorption in the DCT, and the respective inhibitory, kidney-specific kinase-defective KS-WNK1 were expressed at lower levels in the cKO mice. Taken together, these data suggest specific compensatory alterations in components of both paracellular and transcellular renal ion transport mechanisms in mice deficient in claudin-10 in the TAL.

Urinalysis demonstrated that the inhibition of TAL tubular transport by furosemide resulted in a completely differ­ent pattern of tubular Ca2+ and Mg2+ handling that identifies the TAL as the major nephron segment affected by claudin-10 deficiency.  Interestingly, the different effects on plasma Mg2+ and Ca2+ levels reflect the different major reabsorption sites of these ions. Some 60% of the filtered Mg2+ is reabsorbed in the TAL, compared with only 20% of the filtered Ca2+ load (20). Ca2+ hyperreabsorption in TAL seems to be balanced by reduced (proximal and) distal tubular Ca2+ transport. The hyperreabsorption of divalent cations in mice deficient in claudin-10 is in opposition to the loss of divalent cations seen in mouse models of claudin-16 deficiency and in human patients with mutation in CLDN16 or CLDN19. This finding indicates that claudins in the TAL have functions that differentially affect paracellular cation transport in this segment. Mice deficient for claudin-10b in the TAL exhibit decreased permeability for Na+ and increased permeability for Ca2+ and Mg2+, whereas in mice with claudin-16 or claudin-19 deficiency, decreased sodium per­meability in the TAL is paralleled by decreased reabsorption of Ca2+ and Mg2+.

1. Greger R (1981) Cation selectivity of the isolated perfused cortical thick ascending limb of Henle’s loop of rabbit kidney. Pflugers Arch 390:30–37.
2. Furuse M (2010) Molecular basis of the core structure of tight junctions. Cold Spring Harb Perspect Biol 2:a002907.
3. Konrad M, et al. (2006) Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular in­volvement. Am J Hum Genet 79:949–957.
4.  Simon DB, et al. (1999) Paracellin-1, a renal tight junction protein required for par-acellular Mg2+ resorption. Science 285:103–106.
5. Hou J, et al. (2007) Transgenic RNAi depletion of claudin-16 and the renal handling of magnesium. J Biol Chem 282:17114–17122.
6. Himmerkus N, et al. (2008) Salt and acid-base metabolism in claudin-16 knockdown mice: Impact for the pathophysiology of FHHNC patients. Am J Physiol Renal Physiol 295:F1641–F1647.

 PMID: 22891322  PMCID: PMC3435183   http://www.ncbi.nlm.nih.gov/pubmed/22891322

Paracellin-1 is critical for magnesium and calcium reabsorption in the human thick ascending limb of Henle.

Blanchard A, Jeunemaitre X, Coudol P, Dechaux M, Froissart M, et al.
Université Pierre et Marie Curie, INSERM and Laboratoire de Génétique Moléculaire, Hôpital Universitaire Européen Georges Pompidou, Paris, France. blanch@ccr.jussieu.fr
Kidney Int. 2001 Jun; 59(6):2206-15.

A new protein, named paracellin 1 (PCLN-1), expressed in human thick ascending limb (TAL) tight junctions, possibly plays a critical role in the control of magnesium and calcium reabsorption, since mutations of PCLN-1 are present in the hypomagnesemia hypercalciuria syndrome (HHS).
No functional experiments have demonstrated that TAL magnesium and calcium reabsorption were actually impaired in patients with HHS.
Genetic studies were performed in the kindred of two unrelated patients with HHS.

We found two yet undescribed mutations of PCLN-1 (Gly 162 Val, Ala 139 Val). In patients with HHS, renal magnesium and calcium reabsorptions were impaired as expected; NaCl renal conservation during NaCl deprivation and NaCl tubular reabsorption in diluting segment were intact. Furosemide infusion in CS markedly increased NaCl, Mg, and Ca urinary excretion rates. In HHS patients, furosemide similarly increased NaCl excretion, but failed to increase Mg and Ca excretion. Acute MgCl(2) infusion in CS and ERH patient provoked a dramatic increase in urinary calcium excretion without change in NaCl excretion. When combined with MgCl(2) infusion, furosemide infusion remained able to induce normal natriuretic response, but was unable to increase urinary magnesium and calcium excretion further. In HHS patients, calciuric response to MgCl(2) infusion was blunted.

In patients with HHS, levels of circulating renin and aldosterone were normal, suggesting normal blood and extracellular volume. In addition, HHS patient 2 was normally able to lower her sodium excretion below 10 mmol/day during sodium deprivation, and in HHS pa­tient 1, sodium reabsorption in the diluting segment was normal as assessed by hypotonic saline infusion.  After oral NH4Cl load: Minimal urinary pH was 5.8 (normal value <5.4), and maximal net acid excretion reached only 24 pmol/min (normal value >80). Both subjects had hypocitraturia. The latter data suggested in the two probands distal defect of urinary acidification, probably related to nephrocalcinosis.

Because the filtered load of calcium but not the filtered load of magnesium remains unchanged during acute magnesium infusion in humans, the increase in calcium excretion is a better index of the inhibitory effect of peritubular magnesium on renal tubular divalent cation transport.  Urinary sodium excretion remained almost constant in both subjects during MgCl2 infusion (data not shown). Accordingly, the FECa/FENa ratio, which should remain constant if sodium reabsorption was primarily affected, increased in the CS and EHR patient.  Before the furosemide infusion, serum ultrafilterable (UF) Ca concentrations were similar in patients with HHS and the controls. However, Ca excretion markedly differed and was approximately five times higher in HHS patients than in controls.

In the two patients with homozygous mutations in the PCLN-1 gene, an impairment in renal tubular magne­sium and calcium reabsorption with normal NaCl recla­recla­mation was demonstrated. Accordingly, comparative studies performed under baseline condition in one pa­tient with ERH and in HHS patients demonstrated that the magnesium and calcium excretion in HHS patients were inappropriately high when compared with serum magnesium and calcium concentrations. However, renal NaCl reabsorption in HHS patients was intact. There was no clinical evidence of extracellular fluid volume  contraction. Furthermore, basal circulating renin and aldosterone concentrations were normal and adapted to the normal Na intake. Finally, abnormal NaCl reclama­tion in the diluting segment of the nephron was excluded in one patient, while the other was able to adapt normally to a sodium deprived diet.

This study is the first to our knowledge to demonstrate that homozygous mutations of PCLN-1 result in a selective defect in paracellular Mg and Ca reabsorption in the TAL, with intact NaCl reabsorption ability at this site. In addition, the study supports a selective physiological effect of basolateral Mg(2+) and Ca(2+) concentration on TAL divalent cation paracellular permeability, that is, PCLN-1 activity.   PMID: 11380823   http://www.ncbi.nlm.nih.gov/pubmed/11380823

Development of a Novel Sodium-Hydrogen Exchanger Inhibitor for Heart Failure

Elizabeth Juneman*, Reza Arsanjani, Hoang M Thai, Jordan Lancaster, Jeffrey B Madwed, Steven Goldman
Citation: Elizabeth Juneman, et al. (2013) Development of a Novel Sodium-Hydrogen Exchanger Inhibitor for Heart Failure. J Cardio Vasc Med 1: 1-6

This study was designed to determine the potential therapeutic effects of a new sodium-hydrogen exchanger (NHE-1) inhibitor in the rat coronary artery ligation model of chronic heart failure. After the induction of acute myocardial infarction, rats were entered randomly dose dranging from 0.3 mg/kg, 1.0 mg/kg, and 3.0 mg/kg. Solid state micrometer hemodynamics, echocardiographic, and pressure-volume relationships were measured after 6 weeks of treatment. Treatment with this NHE- 1 inhibitor at 3 mg/kg increased (P< 0.05) ejection fraction from 23±3% (N=6) to 33±2% (N=13) while the 1 mg/kg dose decreased (P< 0.05) the infarct size in CHF rats from 21.7±1.4% (N=7) to 15.9±0.7% (N=3) and prevented (P< 0.05) dilatation of the left ventricle in CHF rats in diastole (1.0±0.1 cm, N=6) to 0.9±0.1 cm, N=10) and in systole (0.9±0.1 cm, N=6) to 0.8±0.1, N=10). These study results suggest that this new NHE-1 inhibitor may be potentially useful in treating CHF with an improvement in maladaptive left ventricule remodeling. Because the mechanism of action of this agent is entirely different than the currently applied approach in treating CHF that focuses on aggressive neurohormonal blockade and because this agent does not adversely affect important hemodynamic variables, further investigations with this agent may be warranted.

Keywords: Congestive heart failure; Sodium/hydrogen exchange; Cardiovascular disease; Cardiovascular drugs; CHF: Chronic Heart Failure; NHE-1: Sodium-Hydrogen Exchanger; NCX: Sodium-Calcium Exchanger; Ca2+: Calcium; Na+: Sodium; Na+-K+ATPase: Sodium-Potassium ATPase; NKCC: Sodium-Potassium-Chloride co-transporter; MI: Myocardial Infarction; BI: Boehringer Ingelheim; LV: left Ventricle; EF: Ejection Fraction; LVD: left ventricular dysfunction; PV: Pressure-Volume; SE: Standard Error; ARB: Angiotensin Receptor Blocker; ACE: Angiotensin Converting Enzyme

Without reviewing the pathophysiology of CHF here, altered calcium (Ca2+) handling is a hallmark of CHF. Intracellular Ca2+ concentration is closely regulated by sodium-calcium exchanger (NCX) and Ca2+ efflux is dependent on the intracellular sodium (Na+) concentration and trans-sarcolemmal Na gradient. Multiple channels including sodium-potassium ATPase (Na+-K+ ATPase), sodium-hydrogen exporter (NHE), sodium-bicarbonate co-transporter, sodium-potassium-chloride co-transporter (NKCC), and sodium-magnesium exchanger are responsible for regulation of intracellular sodium in cardiac myocytes. The intracellular concentration of Na+ is significantly increased in heart failure, primarily due to influx of Na+. The NHE plays an integral part in rise of intracellular Na+ concentration and development of hypertrophy in heart failure. Because of its multifaceted role in myocardial function, there has been interest in examining the effects of NHE-1 inhibitors in heart failure.

In this study we report the physiologic responses of a new NHE-1 inhibitor, in a rodent model of heart failure. Previous evaluation of the pharmacokinetic properties of this agent in rat and dog revealed low clearance and robust oral bioavailability, suggesting a potential for once daily oral administration. This new compound was found to be potentially effective in preventing ischemic injury in isolated cells systems and in ischemic injury in isolated cells systems and in a Langendorff isolated heart preparation. Based on these encouraging a pharmacokinetic data, and the established preclinical roof of principle, the next step in new drug development was to test this inhibitor in an appropriate disease-relevant animal model. For this, we chose the rat coronary ligation model of CHF, which is the established model of chronic ischemic heart failure and well performed in our laboratory. The model with permanent occlusion of the left coronary artery is important because this model a similar to the clinical syndrome of CHF. This rat coronary artery model of CHF is the same model used in the classic study defining the beneficial use of angiotensin converting enzyme inhibition with captopril in the treatment of CHF. Thus results in this model have the potential to be predictive of the clinical response seen in patients.

Results

In vivohemodynamic effect of NHE1: As noted previously by our laboratory, rats with severe CHF compared to Sham had changes (P< 0.05) in right ventricular weight, mean arterial pressure, tau, the time constant of LV relaxation, LV systolic pressure, LV end-diastolic pressure, +LVdP/dt, -LVdP/dt, dead volume and peak developed pressure. In this study, treatment resulted in no changes in body weight, chamber weight or hemodynamics. Because we stopped the lowest dose (0.3 mg/kg) there are only hemodynamic data with this dose in rats with CHF.

Echocardiographic changes in LV function and Dimensions with NHE1: Rats with CHF have decreases in EF accompanied by increases in LV systolic and diastolic dimensions. There was no change in anterior wallsystolic displacement. These data are consistent with other reports in this model showing that at 6 weeks after left coronary artery ligation, rats with large MIs have dilated left ventricles with LV remodeling and poor LV function (14,15). Treatment with the highest dose of 3 mg/kg increased (P< 0.05) ejection fraction from 23±3% (N=6) to 33±2% (N=13). Treatment with 1 mg/kg prevented maladaptive LV remodeling, it prevented (P< 0.05) dilatation of the LV in CHF rats in diastole (1.0±0.1 cm, N=6) to 0.9±0.1 cm, N=10) and in systole (0.9±0.1 cm, N=6) to 0.8±0.1, N=10) with no change anterior wall thickening.

Pressure-Volume relationships:  Although there are no significant changes in the PV relationships for either the Sham or CHF rats, there is a trend for the PV loop in CHF to be shifted toward the pressure axis with treatment. These data are consistent with the trend toward decreases in LV dimensions seen with treatment in CHF rats.

Discussion

This study can be viewed as a corollary of a pilot Phase II clinical trial to look for a signal of a beneficial physiologic effect of this new NHE-1 inhibitor in CHF. In terms of drug development, this is an appropriate approach, i.e., take an agent with a therapeutic focus, with an acceptable toxicology profile, alter its pharmacokinetics to improve its oral delivery and bioavailability and then study the drug in an appropriate animal model. The administration of this agent to rats with CHF after left coronary artery ligation resulted in a therapeutic benefit with an increase in EF and a decrease in infarct size in rats with the largest infarcts. There is a suggestion of the prevention of LV remodeling with decreases in LV end-diastolic and end-systolic dimensions accompanied by a similar trend in the PV loop with a shift toward the pressure axis. There were no changes in hemodynamics.

Importantly, the decrease in infarct size with no changes in hemodynamicswould positively affect LV remodeling by minimizing LV dilatation without changes in LV afterload. From a therapeutic perspective, an agent like this may be advantageous in the treatment of heart failure after MI. The lack of hemodynamic changes is not a clinical problem because we already have agents that decrease afterload and lower LV end-diastolic pressure such as angiotensin converting enzyme (ACE) inhibitors and angiotens in receptor blockers (ARBs). In treating CHF, we also have diuretics to control blood volume, which in turn reduces LV end-diastolic pressure. The other potential advantage of an NHE-1 inhibitor is that as opposed to our current use of aggressive neurohormonal blockade, this represents a different approach to treating CHF. This is attractive because we essentially have exhausted or maximized our effects of neurohumoral blockade and with no real new treatments for CHF introduced in the last 10-15 years, need to look for other approaches to treat CHF.

Drug development is obviously a complicated and expensive undertaking. In exploring this agent, we would proposea stepwise approach. In this case with an agent whose analogs have been studied extensively, our thought would be to perform a larger dose ranging study in CHF rats to define dose response curves for systolic function as well as obtain more information on pharmacokinetics, as well as diastolic function and structural changes.

An attractive aspect of this work is that we are examining an agent with a different mechanisms of action that current treatments for heart failure. The stimulus to study the agent in an animal model of heart failure was based on multifaceted roles of sodium-hydrogen exchangers on myocardial function. Nine isoforms of NHE have currently been identified, with NHE- 1 being the predominant isoform in the plasma membrane of the myocardium [3,24]. Because NHE is activated by intracellular acidosis, angiotensin II, and catecholamines, its activity is expectedly increased in heart failure. Inhibition of NHE-1 has previously been associated with decreased fibrosis, apoptosis, preserved contractility, and attenuation of hypertrophy and development of heart failure.

1. Baartscheer A, van Borren MMGJ (2008) Sodium Ion Transporters as New Therapeutic Targets in Heart Failure. Cardiovasc Hematol Agents Med Chem 6: 229-236.
2. Murphy E, Eisner DA (2009) Regulation of Intracellular and Mitochondrial Sodium in Health and Disease. Circ Res 104: 292-303
3. Despa S, Islam MA, Weber CR, Pogwizd SM, Bers DM (2002) Intracellular Na(+) Concentration is Elevated in Heart Failure but Na/K Pump Function is Unchanged. Circulation 105: 2543-2548.
4. Baartscheer A, Schumacher CA, van Borren MMGJ, Belterman CNW, Coronel R, et al. (2003) Increased Na+/H+-Exchange Activity is the Cause of Increased [Na+]i and Underlies Disturbed Calcium Handling in the Rabbit Pressure and Volume Overload Heart Failure Model. Cardiovasc Res 57: 1015-1024.
5.  Pieske B, Houser SR (2003) [Na+]i Handling in the Failing Human Heart. Cardiovasc Res 57: 874-886.
6.  Engelhardt S, Hein L, Keller U, Klämbt K, Lohse MJ (2002) Inhibition of Na(+)-H(+) Exchange Prevents Hypertrophy, Fibrosis, and Heart Failure in Beta(1)- Adrenergic Receptor Transgenic Mice. Circ Res 90: 814-819.
7.  Chen L, Chen CX, Gan XT, Beier N, Scholz W, et al. (2004) Inhibition and Reversal of Myocardial Infarction-Induced Hypertrophy and Heart Failure by NHE-1 Inhibition. Am J Physiol Heart Circ Physiol 286: 381-387.
8.  Marano G, Vergari A, Catalano L, Gaudi S, Palazzesi S, et al. (2004) Na+/ H+ Exchange Inhibition Attenuates Left Ventricular Remodeling and Preserves Systolic Function in Pressure-Overloaded Hearts. Br J Pharmacol 141: 526-532.
9. Goldman S, Raya TE (1995) Rat Infarct Model of Myocardial Infarction and Heart Failure. J Card Fail 1: 169-177.
10. Gaballa MA, Goldman S (2002) Ventricular Remodeling in Heart Failure. J Card Fail. 8: 476-485.
11. Pfeffer MA, Pfeffer JM, Steinberg C, Finn P (1985) Survival After Experimental Myocardial Infarction: Beneficial Effects of Long-Term Therapy with Captopril. Circulation 72: 406-412.
12. Raya TE, Gay RG, Aguirre M, Goldman S (1989) Importance of Venodilatation in Prevention of Left Ventricular Dilatation after Chronic Large Myocardial Infarction in Rats: A Comparison of Captopril and Hydralazine. Circ Res 64: 330-337.

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Selective Ion Conduction

Reviewer and Curator: Larry H Bernstein, MD, FCAP

Introduction

This is Part III of a series of articles on Translational Medicine in water transport or paracellelar flow, and ion conductance.  The first article was solely on the aquaporins (Part I), and the second goes from paracellular flow (dealing with paracellin-1 and the familay of claudins.  These proteins factor into a number of diseases of kidney function and Mg(2+) homeostasis, as well as a relationship between congestive heart failure related to infarct remodeling and the sodium-calcium transporter, with a model for treatment (Part II).  The last explores the basis of selective ion conduction, based on the 2003 Nobel Prize presentation by Roderick MacKinnon (Part III).

POTASSIUM CHANNELS AND THE ATOMIC BASIS OF SELECTIVE ION CONDUCTION

Nobel Lecture, December 8, 2003
Roderick MacKinnon
Howard Hughes Medical Institute, Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University ,NewYork, NY 

Water is an electrically polarizable substance, which means that its molecules rearrange in an ion’s electric field, pointing negative oxygen atoms in the direction of cations and positive hydrogen atoms toward anions. These electrically stabilizing interac­tions are much weaker in a less polarizable substance such as oil. Thus, an ion will tend to stay in the water on either side of a cell membrane rather than en­ter and cross the membrane. And yet numerous cellular processes, ranging from electrolyte transport across epithelia to electrical signal production in neurons, depend on the flow of ions across the membrane. To mediate the flow, specific protein catalysts known as ion channels exist in the cell mem­brane. Ion channels exhibit the following three essential properties: (1) they conduct ions rapidly, (2) many ion channels are highly selective, meaning only certain ion species flow while others are excluded, (3) their function is regu­lated by processes known as gating, that is, ion conduction is turned on and off in response to specific environmental stimuli. Figure 1 summarizes these properties (figure 1).

MacKinnon. Fig 1  Ion channels exhibit three basic properties

Figure 1. Ion channels exhibit three basic properties depicted in the cartoon. They conduct specific ions (for example K ) at high rates, they are selective (a K  channel essentially excludes Na ), and conduction is turned on and off by opening and closing a gate, which can be regulated by an external stimulus such as ligand-binding or membrane voltage. The relative size of K and Naions is shown.

The modern history of ion channels began in 1952 when Hodgkin and Huxley published their seminal papers on the theory of the action potential in the squid giant axon (Hodgkin and Huxley, 1952a; Hodgkin and Huxley, 1952b; Hodgkin and Huxley, 1952c; Hodgkin and Huxley, 1952d). A funda­mental element of their theory was that the axon membrane undergoes changes in its permeability to Na+ and K+ ions. The Hodgkin-Huxley theory did not address the mechanism by which the membrane permeability changes occur: ions could potentially cross the membrane through channels or by a carrier-mediated mechanism. In their words ‘Details of the mecha­nism will probably not be settled for some time’ (Hodgkin and Huxley, 1952a). It is fair to say that the pursuit of this statement has accounted for much ion channel research over the past fifty years.

As early as 1955 experimental evidence for channel mediated ion flow was obtained when Hodgkin and Keynes measured the directional flow of K+ ions across axon membranes using the isotope 42K+ (Hodgkin and Keynes, 1955). They observed that K+ flow in one direction across the membrane depends on flow in the opposite direction, and suggested that ‘the ions should be con­strained to move in single file and that there should, on average, be several ions in a channel at any moment’. Over the following two decades Armstrong and Hille used electrophysiological methods to demonstrate that Na+ and K+ ions cross cell membranes through unique protein pores – Na+ channels and K+ channels – and developed the concepts of selectivity filter for ion discrim­ination and gate for regulating ion flow (Hille, 1970; Hille, 1971; Hille, 1973; Armstrong, 1971; Armstrong et al., 1973; Armstrong and Bezanilla, 1977; Armstrong, 1981). The patch recording technique invented by Neher and Sakmann then revealed the electrical signals from individual ion channels, as well as the extraordinary diversity of ion channels in living cells throughout nature (Neher and Sakmann, 1976).

The past twenty years have been the era of molecular biology for ion chan­nels. The ability to manipulate amino acid sequences and express ion chan­nels at high levels opened up entirely new possibilities for analysis. The ad­vancement of techniques for protein structure determination and the devel­opment of synchrotron facilities also created new possibilities. For me, a sci­entist who became fascinated with understanding the atomic basis of life’s electrical system, there could not have been a more opportune time to enter the field.

The past twenty years have been the era of molecular biology for ion channels. The ability to manipulate amino acid sequences and express ion channels at high levels opened up entirely new possibilities for analysis. The advancement of techniques for protein structure determination and the development of synchrotron facilities also created new possibilities. For me, a scientist who became fascinated with understanding the atomic basis of life’s electrical system, there could not have been a more opportune time to enter the field. 

MY EARLY STUDIES: THE K+ CHANNEL SIGNATURE SEQUENCE

The cloning of the Shaker K+ channel gene from Drosophila melanogaster by Jan, Tanouye, and Pongs revealed for the first time a K+ channel amino acid se­quence and stimulated efforts by many laboratories to discover which of these amino acids form the pore, selectivity filter, and gate (Tempel et al., 1987; Kamb et al., 1987; Pongs et al., 1988). At Brandeis University in Chris Miller’s laboratory I had an approach to find the pore amino acids. Chris and I had just completed a study showing that charybdotoxin, a small protein from scor­pion venom, inhibits a K+ channel isolated from skeletal muscle cells by plug­ging the pore and obstructing the flow of ions (MacKinnon and Miller, 1988). In one of those late night ‘let’s see what happens if’ experiments while taking a molecular biology course at Cold Spring Harbor I found that the toxin – or what turned out to be a variant of it present in the charybdotoxin prepara­tion – inhibited the Shaker K+ channel (MacKinnon et al., 1988; Garcia et al., 1994). This observation meant I could use the toxin to find the pore, and it did not take very long to identify the first site-directed mutants of the Shaker K+ channel with altered binding of toxin (MacKinnon and Miller, 1989). I continued these experiments at Harvard Medical School where I began as as­sistant professor in 1989. Working with my small group at Harvard, including Tatiana Abramson, Lise Heginbotham, and Zhe Lu, and sometimes with Gary Yellen at Johns Hopkins University, we reached several interesting conclusions concerning the architecture of K+ channels. They had to be tetramers in which four subunits encircle a central ion pathway (MacKinnon, 1991). This conclusion was not terribly surprising but the experiments and analysis to reach it gave me great pleasure since they required only simple measure­ments and clear reasoning with binomial statistics. We also deduced that each subunit presents a ‘pore loop’ to the central ion pathway (figure 2) (MacKinnon, 1995).

MacKinnon. Fig 2.  tetramer K channel

Figure 2. Early picture of a tetramer K+ channel with a selectivity filter made of pore loops. A linear representation of a Shaker K+ channel subunit on top shows shaded hydrophobic segments S1 to S6 and a region designated the pore loop. A partial amino acid sequence from the Shaker K+ channel pore loop highlights amino acids shown to interact with ex-tracellular scorpion toxins (*), intracellular tetraethylammonium (↑) and K+ ions (+). The pore loop was proposed to reach into the membrane (middle) and form a selectivity filter at the center of four subunits (bottom).

This ‘loop’ formed the binding sites for scorpion toxins (MacKinnon and Miller, 1989; Hidalgo and MacKinnon, 1995; Ranganathan et al., 1996) as well as the small-molecule inhibitor tetraethylammonium ion (MacKinnon and Yellen, 1990; Yellen et al., 1991), which had been used by Armstrong and Hille decades earlier in their pioneering analysis of K+ channels (Armstrong, 1971; Armstrong and Hille, 1972). Most important to my thinking, mutations of certain amino acids within the ‘loop’ affected the channel’s ability to discriminate between K+ and Na+, the selectivity hallmark of K+ channels (Heginbotham et al., 1992; Heginbotham et al., 1994). Meanwhile, new K+ channel genes were discovered and they all had one ob­vious feature in common: the very amino acids that we had found to be im­portant for K+ selectivity were conserved (figure 3). We called these amino acids the K+ channel signature sequence, and imagined four pore loops somehow forming a selectivity filter with the signature sequence amino acids inside the pore (Heginbotham et al., 1994; MacKinnon, 1995).

Figure 3. The K+ channel signature sequence shown as single letter amino acid code (blue) is highly conserved in organisms throughout the tree of life. Some K+ channels contain six membrane-spanning segments per subunit (6TM) while others contain only two (2TM). 2TM K+ channels correspond to 6TM K+ channels without the first four membrane-span­ning segments (S1-S4 in figure 2).

When you consider the single channel conductance of many K+ channels found in cells you realize just how incredible these molecular devices are. With typical cellular electrochemical gradients, K+ ions conduct at a rate of 107 to 108 ions per second. That rate approaches the expected collision fre­quency of K+ ions from solution with the entryway to the pore. This means that K+ ions flow through the pore almost as fast as they diffuse up to it. For this to occur the energetic barriers in the channel have to be very low, some­thing like those encountered by K+ ions diffusing through water. All the more remarkable, the high rates are achieved in the setting of exquisite selectivity: the K+ channel conducts K+, a monovalent cation of Pauling radius 1.33 Å, while essentially excluding Na+, a monovalent cation of Pauling radius 0.95 Å. And this ion selectivity is critical to the survival of a cell. How does nature ac­complish high conduction rates and high selectivity at the same time? The an­swer to this question would require knowing the atomic structure formed by the signature sequence amino acids, that much was clear. The conservation of the signature sequence amino acids in K+ channels throughout the tree of life, from bacteria (Milkman, 1994) to higher eukaryotic cells, implied that nature had settled upon a very special solution to achieve rapid, selective K+ conduction across the cell membrane. For me, this realization provided in­spiration to want to directly visualize a K+ channel and its selectivity filter.

THE KCSA STRUCTURE AND SELECTIVE K+ CONDUCTION

I did not know how we would ever reach the point of ob­taining enough K+ channel protein to attempt crystallization, but the K+ channel signature sequence continued to appear in a growing number of prokaryotic genes, making expression in Escherichia coli possible. We focused our effort on a bacterial K+ channel called KcsA from Streptomyces lividans, dis­covered by Schrempf (Schrempf et al., 1995). The KcsA channel has a simple topology with only two membrane spanning segments per subunit corre­sponding to the Shaker K+ channel without S1 through S4 (figure 2). Despite its prokaryotic origin KcsA closely resembled the Shaker K+ channel’s pore amino acid sequence, and even exhibited many of its pharmacological prop­erties, including inhibition by scorpion toxins (MacKinnon et al., 1998). This surprised us from an evolutionary standpoint, because why should a scorpion want to inhibit a bacterial K+ channel! But from the utilitarian point of view of protein biophysicists we knew exactly what the scorpion toxin sensitivity meant, that KcsA had to be very similar in structure to the Shaker K+ channel.

The KcsA channel produced crystals but they were poorly ordered and not very useful in the X-ray beam. After we struggled for quite a while I began to wonder whether some part of the channel was intrinsically disordered and in­terfering with crystallization. Fortunately my neighbor Brian Chait and his postdoctoral colleague Steve Cohen were experts in the analysis of soluble proteins by limited proteolysis and mass spectrometry, and their techniques applied beautifully to a membrane protein. We found that KcsA was as solid as a rock, except for its C-terminus. After removing disordered amino acids from the c-terminus with chymotrypsin the crystals improved dramatically, and we were able to solve an initial structure at a resolution of 3.2 Å (Doyle et al., 1998). We could not clearly see K+ in the pore at this resolution, but my years of work on K+ channel function told me that Rb+ and Cs+ should be valuable electron dense substitutes for K+, and they were. Rubidium and Cs+ difference Fourier maps showed these ions lined up in the pore – as Hodgkin and Keynes might have imagined in 1955 (Hodgkin and Keynes, 1955).

The KcsA structure was altogether illuminating, but before I describe it, I will depart from chronology to explain the next important technical step. A very accurate description of the ion coordination chemistry inside the selec­tivity filter would require a higher resolution structure. With 3.2 Å data we could infer the positions of the main-chain carbonyl oxygen atoms by apply­ing our knowledge of small molecule structures, that is our intu­ition, but we needed to see the selectivity filter atoms in detail. A high-resolu­tion structure was actually quite difficult to obtain. After more than three ad­ditional years of work by João and then Yufeng (Fenny) Zhou we finally man­aged to produce high-quality crystals by attaching monoclonal Fab fragments to KcsA. These crystals provided the information we needed, a structure at a resolution of 2.0 Å in which K+ ions could be visualized in the grasp of selectivity filter protein atoms (figure 4) (Zhou et al., 2001b). What did the K+ channel structure tell us and why did nature conserve the K+ channel signa­ture sequence amino acids?

MacKinnon Fig 4. Electron density KcsA K channel

Figure 4. Electron density (2Fo-Fc contoured at 2 ir) from a high-resolution structure of the KcsA K+ channel is shown as blue mesh. This region of the channel features the selectivity filter with K+ ions and water molecules along the ion pathway. The refined atomic model is shown in the electron density. Adapted from (Zhou et al., 2001b).

Not all protein structures speak to you in an understandable language, but the KcsA K+ channel does. Four subunits surround a central ion pathway that crosses the membrane (figure 5A). Two of the four subunits are shown in fig­ure 5B with electron density from K+ ions and water along the pore. Near the center of the membrane the ion pathway is very wide, forming a cavity about 10 Å in diameter with a hydrated K+ ion at its center. Each subunit directs the C-terminal end of a ‘pore helix’, shown in red, toward the ion. The C-termi­nal end of an á-helix is associated with a negative ‘end charge’ due to car­bonyl oxygen atoms that do not participate in secondary structure hydrogen bonding, so the pore helices are directed as if to stabilize the K+ ion in the cavity. At the beginning of this lecture I raised the fundamental issue of the cell membrane being an energetic barrier to ion flow because of its oily inte­rior. KcsA allows us to intuit a simple logic encoded in its structure, and elec­trostatic calculations support the intuition (Roux and MacKinnon, 1999): the K+ channel lowers the membrane dielectric barrier by hydrating a K+ ion deep inside the membrane, and by stabilizing it with á-helix end charges.

MacKinnon Fig 5.  KcsA K+ channel   pore-helices (red) and selectivity filter (yellow)

Figure 5. (A) A ribbon representation of the KcsA K+ channel with its four subunits colored uniquely. The channel is oriented with the extracellular solution on top. (B) The KcsA K+ channel with front and back subunits removed, colored to highlight the selectivity filter (yellow). Electron density in blue mesh is shown along the ion pathway. Labels identify the pore, outer, and inner helices and the inner helix bundle. The outer and inner helices correspond to S5 and S6 in figure 2

How does the K+ channel distinguish K+ from Na+? Our earlier mutagene-sis studies had indicated that the signature sequence amino acids would be re­sponsible for this most basic function of a K+ channel. Figure 6 shows the structure formed by the signature sequence – the selectivity filter – located in the extracellular third of the ion pathway. The glycine amino acids in the se­quence TVGYG have dihedral angles in or near the left-handed helical region of the Ramachandran plot, as does the threonine, allowing the main-chain carbonyl oxygen atoms to point in one direction, toward the ions along the pore. It is easy to understand why this sequence is so conserved among K+ channels: the alternating glycine amino acids permit the required dihedral angles, the threonine hydroxyl oxygen atom coordinates a K+ ion, and the side-chains of valine and tyrosine are directed into the protein core sur­rounding the filter to impose geometric constraint.

MacKinnon Figure 6. Detailed structure of the K+ selectivity filter

Figure 6. Detailed structure of the K+ selectivity filter (two subunits). Oxygen atoms coordi­nate K+ ions (green spheres) at positions 1 to 4 from the extracellular side. Single letter amino acid code identifies select signature sequence amino acids. Yellow, blue and red cor­respond to carbon, nitrogen and oxygen atoms, respectively. Green and gray dashed lines show oxygen-K+ and hydrogen bonding interactions.

The end result when the subunits come together is a narrow tube consisting of four equal spaced K+ binding sites, labeled 1 to 4 from the extracellular side. Each binding site is a cage formed by eight oxygen atoms on the vertices of a cube, or a twisted cube called a square antiprism (figure 7). The binding sites are very similar to the single alkali metal site in nonactin, a K+ selective antibiotic with nearly identical K+-oxygen distances (Dobler et al., 1969; Dunitz and Dobler, 1977). The principle of K+ selectivity is implied in a subtle feature of the KcsA crystal structure. The oxygen atoms surrounding K+ ions in the selectivity filter are arranged quite like the water molecules surrounding the hydrated K+ ion in the cavity. This comparison conveys a visual impression of binding sites in the filter paying for the energetic cost of K+ dehydration. The Na+ ion is appar­ently too small for these K+-sized binding sites, so its dehydration energy is not compensated.

MacKinnon Fig 7 K+ channel mimics the hydration shell surrounding a K+ ion

Figure 7. A K+ channel mimics the hydration shell surrounding a K+ ion. Electron density (blue mesh) for K+ ions in the filter and for a K+ ion and water molecules in the central cav­ity are shown. White lines highlight the coordination geometry of K+ in the filter and in wa­ter. Adapted from (Zhou et al., 2001b).

The question that compelled us most after seeing the structure was exactly how many ions are in the selectivity filter at a given time? To begin to under­stand how ions move through the filter we needed to know the stoichiometry of the ion conduction reaction, and that meant knowing how many ions can occupy the filter. Four binding sites were apparent, but are they all occupied at once? Four K+ ions in a row separated by an average center-to-center dis­tance of 3.3 Å seemed unlikely for electrostatic reasons. From an early stage we suspected that the correct number would be closer to two, because two ions more easily explained the electron density we observed for the larger al­kali metal cations Rb+ and Cs+ (Doyle et al., 1998; Morais-Cabral et al., 2001). Quantitative evidence for the precise number of ions came with the high-res­olution structure and with the analysis of Tl+ (Zhou and MacKinnon, 2003). Thallium is the most ideally suited ‘K+ analog’ because it flows through K+ channels, has a radius and dehydration energy very close to K+, and has the favorable crystallographic attributes of high electron density and an anom­alous signal. The one serious difficulty in working with Tl+ is its insolubility with Cl. Fenny meticulously worked out the experimental conditions and de­termined that on average there are between two and two and a half conduct­ing ions in the filter at once, with an occupancy at each position around one half.

We also observed that if the concentration of K+ (or Tl+) bathing the crys­tals is lowered sufficiently (below normal intracellular levels) then a reduc­tion in the number of ions from two to one occurs and is associated with a structural change to a ‘collapsed’ filter conformation, which is pinched closed in the middle (Zhou et al., 2001b; Zhou and MacKinnon, 2003). At concentrations above 20 mM the entry of a second K+ ion drives the filter to a ‘conductive’ conformation, as shown in figure 8. Sodium on the other hand does not drive the filter to a ‘conductive’ conformation even at concentra­tions up to 500 mM.

MacKinnon Figure 8. The selectivity filter can adopt two conformations

Figure 8. The selectivity filter can adopt two conformations. At low concentrations of K+ on average one K+ ion resides at either of two sites near the ends of the filter, which is col­lapsed in the middle. At high concentrations of K+ a second ion enters the filter as it changes to a conductive conformation. On average, two K+ ions in the conductive filter re­side at four sites, each with about half occupancy.

The K+-induced conformational change has thermodynamic consequences for the affinity of two K+ ions in the ‘conductive’ filter. It implies that a frac­tion of the second ion’s binding energy must be expended as work to bring about the filter’s conformational change, and as a result the two ions will bind with reduced affinity. To understand this statement at an intuitive level, rec­ognize that for two ions to reside in the filter they must oppose its tendency to collapse and force one of them out, i.e. the two-ion ‘conductive’ conforma­tion is under some tension, which will tend to lower K+ affinity. This is a de­sirable property for an ion channel because weak binding favors high con­duction rates. The same principle, referred to as the ‘induced fit’ hypothesis, had been proposed decades earlier by enzymologists to explain high speci­ficity with low substrate affinity in enzyme catalysis (Jencks, 1987).

In the ‘conductive’ filter if two K+ ions were randomly distributed then they would occupy four sites in six possible ways. But several lines of evidence hint­ed to us that the ion positions are not random. For example Rb+ and Cs+ ex-hibit preferred positions with obviously low occupancy at position 2 (Morais-Cabral et al., 2001; Zhou and MacKinnon, 2003). In K+ we observed an un­usual doublet peak of electron density at the extracellular entryway to the se­lectivity filter, shown in figure 9 (Zhou et al., 2001b). We could explain this density if K+ is attracted from solution by the negative protein surface charge near the entryway and at the same time repelled by K+ ions inside the filter. Two discrete peaks implied two distributions of ions in the filter.

MacKinnon Fig 9  Figure 9. Two K+ ions in the selectivity filter are hypothesized to exist predominantly in two specific configurations 1,3 and 2,4 as shown.

Figure 9. Two K+ ions in the selectivity filter are hypothesized to exist predominantly in two specific configurations 1,3 and 2,4 as shown. K+ ions and water molecules are shown as green and red spheres, respectively. Adapted from (Zhou et al., 2001b).

Discrete configurations of an ion pair suggested a mechanism for ion con­duction (figure 10A) (Morais-Cabral et al., 2001). The K+ ion pair could dif­fuse back and forth between 1,3 and 2,4 configurations (bottom pathway), or alternatively an ion could enter the filter from one side of the membrane as the ion-water queue moves and a K+ exits at the opposite side (the top path­way). Movements would have to be concerted because the filter is no wider than a K+ ion or water molecule. The two paths complete a cycle: in one com­plete cycle each ion moves only a fraction of the total distance through the fil­ter, but the overall electrical effect is to move one charge all the way.

MacKinnon Fig 10 Figure 10. The selectivity filter is represented as five square planes of oxygen atoms.

Figure 10. (A) Through-put cycle for K+ conduction invoking 1,3 and 2,4 configurations. The selectivity filter is represented as five square planes of oxygen atoms. K+ and water are shown as green and red spheres, respectively. (B) Simulated K+ flux around the cycle is graphed as a function of the energy difference between the 1,3 and 2,4 configurations. Adapted from (Morais-Cabral et al., 2001).

A simulation of ions diffusing around the cycle offers a possible explanation: maximum flux is achieved when the energy difference between the 1,3 and 2,4 configurations is zero because that is the condition under which the ‘energy landscape’ for the con­duction cycle is smoothest (figure 10B). The energetic balance between the configurations therefore might reflect the optimization of conduction rate by natural selection (Morais-Cabral et al., 2001). It is not so easy to demonstrate this point experimentally but it is certainly fascinating to ponder.

COMMON STRUCTURAL PRINCIPLES UNDERLIE K+ AND Cl SELECTIVITY

The focus of this lecture is K+ channels, but for a brief interlude I would like to show you a Cl selective transport protein. By comparing a K+ channel and a Cl ‘channel’ we can begin to appreciate familiar themes in nature’s solu­tions to different problems: getting cations and anions across the cell mem­brane. ClC Cl channels are found in many different cell types and are associ­ated with a number of physiological processes that require Cl ion flow across lipid membranes (Jentsch et al., 1999; Maduke et al., 2000). As is the case for K+ channels, ClC family genes are abundant in prokaryotes, a fortunate cir­cumstance for protein expression and structural analysis. When Raimund Dutzler joined my laboratory he, Ernest Campbell and I set out to address the structural basis of Cl ion selectivity. We determined crystal structures of two bacterial members of the ClC Cl channel family, one from Escherichia coli (EcClC) and another from Salmonella typhimurium (StClC) (Dutzler et al., 2002). Recent studies by Miller on the function of EcClC have shown that it is actually a Cl – proton exchanger (Accardi and Miller, 2004). We do not yet know why certain members of this family of Cl transport proteins function as channels and others as exchangers, but the crystal structures are fascinating and give us a view of Cl selectivity. Architecturally the ClC proteins are unre­lated to K+ channels, but if we focus on the ion pathway certain features are similar (figure 11).

MacKinnon Fig 11 CIC Cl transport protein

Figure 11. The overall architecture of K+ channels and ClC Cl transport proteins is very dif­ferent but certain general features are similar. One similarity shown here is the use of á-he-lix end charges directed toward the ion pathway. The negative C-terminal end charge (red) points to K+. The positive N-terminal end charge (blue) points to Cl.

As we saw in K+ channels, the ClC proteins have a-helices pointed at the ion pathway, but the direction is reversed with the positive charge of the N-terminus close to Cl. This makes perfect sense for lowering the dielectric barrier for a Cl ion. In ClC we see that ions in its selectivity fil­ter tend to be coordinated by main chain protein atoms, with amide nitrogen atoms surrounding Cl instead of carbonyl oxygen atoms surrounding K+ (figure 12). We also see that both the K+ and Cl selectivity filters contain multiple close-spaced binding sites and appear to contain more than one ion, perhaps to exploit electrostatic repulsion between ions in the pore. I find these simi­larities fascinating. They tell us that certain basic physical principles are im­portant, such as the use of á-helix end charges to lower the dielectric barrier when ions cross the lipid membrane.

TRYING TO SEE A K+ CHANNEL OPEN AND CLOSE

channels conduct when called upon by a specific stimulus such as the binding of a ligand or a change in membrane voltage (Hille, 2001). The processes by which ion conduction is turned on are called gating. The con­duction of ions occurs on a time scale that is far too rapid to involve very large protein conformational changes.

Figure 12. K+ and Cl- selectivity filters make use of main chain atoms to coordinate ions

Figure 12. K+ and Cl selectivity filters make use of main chain atoms to coordinate ions: car­bonyl oxygen atoms for K+ ions (green spheres) and amide nitrogen atoms for Cl ions (red spheres). Both filters contain multiple close-spaced ion binding sites. The Cl selectivity fil­ter is that of a mutant ClC in which a glutamate amino acid was changed to glutamine (Dutzler et al., 2003).

In the KcsA K+ channel gating is controlled by intracellular pH and lipid membrane composition, but unfortunately the KcsA channel’s open proba­bility reaches a maximum value of only a few percent in functional assays (Cuello et al., 1998; Heginbotham et al., 1998). At first we had no definitive way to know whether a gate was open or closed in the crystal structures. In the 1970s Armstrong had proposed the existence of a gate near the intracel­lular side of the membrane in voltage dependent K+ channels because he could ‘trap’ large organic cations inside the pore between a selectivity filter near the extracellular side and a gate near the intracellular side (Armstrong, 1971; Armstrong, 1974). Following these ideas we crystallized KcsA with a heavy atom version of one of his organic cations, tetrabutyl antimony (TBA), and found that it binds inside the central cavity of KcsA (Zhou et al., 2001a). This was very interesting because the ~10 Å diameter of TBA far exceeds the pore diameter leading up to the cavity: in KcsA the intracellular pore entry­way is constricted to about 3.5 Å by the inner helix bundle (figure 5B). Seeing TBA ‘trapped’ in the cavity behind the inner helix bundle evoked Arm-strong’s classical view of K+ channel gating, and implied that the inner helix bundle serves as a gate and is closed in KcsA. Mutational and spectroscopic studies in other laboratories also pointed to the inner helix bundle as a pos­sible gate-forming structural element (Perozo et al., 1999; del Camino et al., 2000).

We subsequently determined the crystal structure of MthK, complete K+ channel containing RCK domains, from Methanobacterium thermoautotrophicus (figure 13) (Jiang et al., 2002a). This structure was extremely informative. The RCK domains form a ‘gating ring’ on the intracellular side of the pore. In clefts between domains we could see what appeared to be divalent cation binding sites, and the crys­tals had been grown in the presence of Ca2+. In functional assays we discov­ered that the open probability of the MthK channel increased as Ca2+ or Mg2+ concentration was raised, giving us good reason to believe that the crystal structure should represent the open conformation of a K+ channel.

In our MthK structure the inner helix bundle is opened like the aperture of a camera (figure 14) (Jiang et al., 2002b). As a result, the pathway leading up to the selectivity filter from the intracellular side is about 10 Å wide, explaining how Armstrong’s large organic cations can enter the cavity to block a K+ chan-nel, and how K+ ions gain free access to the selectivity filter through aqueous diffusion. By comparing the KcsA and MthK channel structures it seemed that we were looking at examples of closed and opened K+ channels, and could easily imagine the pore undergoing a conformational change from closed to open.

In the crystal of KvAP the voltage sen­sors, held by monoclonal Fab fragments, adopted a non-native conformation. This observation in itself is meaningful as it underscores the intrinsic flexibil­ity of voltage sensors: in contrast Fab fragments had little effect on the more rigid KcsA K+ channel and ClC Cl channel homolog, both of which we deter­mined in the presence and absence of Fab fragments (Doyle et al., 1998; Zhou et al., 2001b; Dutzler et al., 2002; Dutzler et al., 2003). KvAP’s voltage sensors contain a hydrophobic helix-turn-helix element with arginine residues beside the pore (Jiang et al., 2003a).

The KvAP structure and associated functional studies have provided a conceptual model for voltage-dependent gating – one in which the voltage sensors move at the protein-lipid interface in response to a balance between hydrophobic and electrostatic forces. Rees and colleagues at the California Institute of Technology determined the structure of a voltage regulated mechanosensitive channel called MscS, and although it is unrelated to traditional voltage-dependent channels, it too con­tains hydrophobic helix-turn-helix elements with arginine residues apparent­ly against the lipid membrane (Bass et al., 2002). MscS and KvAP are fascinat­ing membrane protein structures. They do not fit into the standard category of membrane proteins with rigid hydrophobic walls against the lipid mem­brane core. I find such proteins intriguing.

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