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Ca2+-Stimulated Exocytosis: The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Posted in Ca2+ triggered activation, Ca2+ triggered activation, Calcium Signaling, Calmodulin, Cell Biology, Signaling & Cell Circuits, Cytoskeleton, Innovations in Neurophysiology & Neuropsychology, Neurohumoral Transmission, PKC, Proteomics, Synaptic vesicle, tagged Ca2+-regulation, Ca2+/calmodulin-dependent protein kinase, Calmodulin, calmodulin-dependent pathway, calmodulin-independent pathway, CaMK, exocytosis, Howard Hughes Medical Institute, PKC, Protein kinase C, SNARE, Stanford University School of Medicine, Synaptic vesicle on December 23, 2013| Leave a Comment »

Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Writer and Curator: Larry H Bernstein, MD, FCAP
and
Curator and Content Editor: Aviva Lev-Ari, PhD, RN

This article is Part V in a series of TWELVE articles, listed at the end of this article,  on the

1. cytoskeleton,
2. calcium calmodulin kinase signaling,
3. muscle and nerve transduction, and
4. calcium,
5. Na+-K+-ATPase,
6. neurohumoral activity and vesicles vital and essential for all functions related to

• cell movement,
• migration, and
• contraction.

Calmodulin and Protein Kinase C Increase Ca–stimulated Secretion by Modulating
Membrane-attached Exocytic Machinery

YA Chen, V Duvvuri, H Schulmani, and RH.Scheller‡

From the ‡Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology,
and the Department of Neurobiology, Stanford University School of Medicine, Stanford, CA

J Biol Chem 1999; 274(37): 25469-26476.

The molecular mechanisms underlying the Ca2+ regulation of hormone and neurotransmitter release
are largely unknown.

Using a reconstituted [3H]norepinephrine release assay in permeabilized PC12 cells, we found

• essential proteins that support the triggering stage of Ca2+-stimulated exocytosis
• are enriched in an EGTA extract of brain membranes.

Fractionation of this extract allowed purification of two factors that stimulate secretion

• in the absence of any other cytosolic proteins.

These are calmodulin and protein kinase Ca (PKCa). Their effects on secretion were

• confirmed using commercial and recombinant proteins.

Calmodulin enhances secretion

• in the absence of ATP, whereas
• PKC requires ATP to increase secretion, suggesting that
• phosphorylation is involved in PKC-mediated stimulation
• but not calmodulin mediated stimulation.
• Both proteins modulate
  • The half-maximal increase was elicited by
    3 nM PKC and 75 nM calmodulin.

These results suggest that calmodulin and PKC increase Ca2+-activated exocytosis by

• directly modulating the membrane- or cytoskeleton-attached exocytic machinery
  downstream of Ca2+ elevation.

The abbreviations used are:

NE, norepinephrine; PKC, protein kinase C; CaM, calmodulin; SNAP-25, synaptosome-associated protein of 25 kDa; CAPS, calcium-dependent activator protein for secretion; SNARE, SNAP (soluble N-ethylmaleimide-sensitive factor attachment proteins) receptor; CaMK, Ca2+/calmodulin-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; AMP-PNP, adenosine 59-(b,g- imido) triphosphate;  HA, hydroxyapatite

*This work was supported in part by Conte Center Grant MH48108. The costs of publication of
this article were defrayed in part by the payment of page charges. This article has been marked
“advertisement” in accordance with 18 U.S.C. Section 1734.

The molecular mechanisms of presynaptic vesicle release have been extensively examined
by a combination of

• biochemical,
• genetic, and
• electrophysiological techniques.

A series of protein-protein interaction cascades have been proposed to lead to vesicle
docking and fusion (1–3). The SNARE protein family, including

• syntaxin, SNAP-25, and vesicle-associated membrane protein
  (VAMP, also called synaptobrevin),
• plays an essential role in promoting membrane fusion, and
• is thought to comprise the basic fusion machinery (4, 5).

In Ca2+-stimulated exocytosis, many additional proteins are important in the Ca2+ regulation
of the basic membrane trafficking apparatus. Calcium

• not only triggers rapid fusion of release-competent vesicles, but is also involved in
• earlier processes which replenish the pool of readily releasable vesicles (6).

Furthermore, it appears to be critical in initiating several forms of synaptic plasticity including

• post-tetanic potentiation (7).

The molecular mechanisms by which Ca2+ regulates these processes is not well understood.

PC12 cells have often been utilized to study Ca2+-activated exocytosis, as

• they offer a homogeneous cell population that possesses the same basic exocytic machinery as neurons (8).

In this study, we used an established cracked cell assay, in which

• [3H]norepinephrine (NE)1 labeled PC12 cells are
• permeabilized by mechanical “cracking” and
• then reconstituted for secretion of NE in the presence of test proteins (9).

Transmitter-filled vesicles and intracellular cytoskeletal structures

• remain intact in these cells,
• while cytosolic proteins leak out (10).

These cracked cells readily release NE upon addition of

• ATP,
• brain cytosol, and
• 1 mM free Ca2+
  • at an elevated temperature.

We term this a “composite assay,” as

• all essential components are added into one reaction mixture.

Alternatively, cracked cells can be

• first primed with cytosol and ATP, washed, then
• reconstituted for NE release with cytosol and Ca2+ (11).

This sequential priming-triggering protocol is useful

• for determining whether a protein acts early or late in the exocytic pathway, and
• whether its effect is dependent on Ca2+ or ATP.

This semi-intact cell system serves as

• a bridge between an in vitro system comprised of purified components, and
• electro-physiological systems that monitor release in vivo.
• It provides information on protein functions in a cell with an intact membrane infrastructure while being easily manipulatable.

Ca2+ regulation by membrane depolarization is no longer a concern, as
intra-cellular Ca2+ concentration can be controlled by a buffered solution.

• Indirect readout of neurotransmitter release using a postsynaptic cell is replaced by
• direct readout of [3H]NE released into the buffer.

Complications associated with interpreting overlapping

• exo- and endocytotic signals are also eliminated as only one round of exocytosis is measured.

Finally, concentration estimates are likely to be accurate, since

• added compounds do not need to diffuse long distances along axons and dendrites to their sites of action.

Using this assay, several proteins required for NE release have been purified from rat brain cytosol, including

• phosphatidyl-inositol transfer protein (12),
• phosphatidylinositol-4-phosphate 5-kinase (13), and
• calcium-dependent activator protein for secretion (CAPS) (9).

The validity of the cracked cell system is confirmed by the finding that

• phosphatidylinositol transfer protein and CAPS are mammalian homologues of
  • yeast SEC14p (12) and
  • nematode UNC31p, respectively (14),
• both proteins involved in membrane trafficking (15, 16).

Calmodulin is the most ubiquitous calcium mediator in eukaryotic cells, yet its involvement in membrane trafficking has not been well established. Some early studies showed

• that calmodulin inhibitors (17–19), anti-calmodulin antibodies (20,21),

or

• calmodulin binding inhibitory peptides (22) inhibited Ca2+-activated exocytosis.

However, in other studies, calmodulin-binding peptides and an anti-calmodulin antibody led to the conclusion that

• calmodulin is only involved in endocytosis,
• not exocytosis (23).

More recently, it was reported that

1. Ca2+/ calmodulin signals the completion of docking and
2. triggers a late step of homotypic vacuole fusion in yeast,

• thus suggesting an essential role for Ca2+/calmodulin in constitutive intracellular membrane fusion (24).

If calmodulin indeed plays an important role in exocytosis,

• a likely target of calmodulin is
• Ca2+/calmodulin-dependent protein kinase II (CaMKII),
  • a multifunctional kinase that is found on synaptic vesicles (25) and
  • has been shown to potentiate neurotransmitter release (26, 27).

Another Ca2+ signaling molecule, PKC, has also been implicated in regulated exocytosis.
In various cell systems, it has been shown that

• the phorbol esters stimulate secretion (28, 29).

It is usually assumed that phorbol esters effect on exocytosis is

• through activation of PKC,
• but Munc13-1 was recently shown to be a presynaptic phorbol ester receptor that enhances neurotransmitter release (30, 31),

which complicates the interpretation of some earlier reports. The mode of action of PKC remains controversial. There is evidence

• that PKC increases the intracellular Ca2+ levels by modulating plasma membrane Ca2+ channels (32, 33),
• that it increases the size of the release competent vesicle pool (34, 35), or
• that it increases the Ca2+ sensitivity of the membrane trafficking apparatus (36).

no consensus on these issues has been reached.

PKC substrates that have been implicated in exocytosis include

1. SNAP-25 (37),
2. synaptotagmin (28),
3. CAPS (38), and
4. nsec1 (39).

It is believed that upon phosphorylation, these PKC substrates might

• interact differently with their binding partners, which, in turn,
• leads to the enhancement of exocytosis.

In addition, evidence is accumulating that PKC and calmodulin interfere with each others actions, as

• PKC phosphorylation sites are embedded in the calmodulin-binding domains of substrates such as
• neuromodulin and
• neurogranin (40).

It is therefore possible that PKC could modulate exocytosis via

• a calmodulin-dependent pathway by synchronously releasing calmodulin from storage proteins.

In this study, we fractionated an EGTA extract of brain membranes in order to identify active components that could reconstitute release in the cracked cell assay system. We identified calmodulin and PKC as two active factors. Thus, we demonstrate that

• calmodulin and PKC play a role in the Ca2+ regulation of exocytosis, and provide further insight into the mechanisms of their action.

DISCUSSION

 In this study, we first identified an EGTA extract of brain membranes as a protein source

•  capable of reconstituting Ca2+- activated exocytosis in cracked PC12 cells.

EGTA only extracts a small pool of Ca2+-dependent membrane-associating proteins,

• it served as an efficient initial purification step.

Further protein chromatography led to the identification of two active factors in the starting extract,

• calmodulin and PKC,
• which together accounted for about half of the starting activity.

Upon confirmation with commercially obtained proteins, this result unambiguously demonstrated

• that calmodulin and PKC mediate aspects of Ca2+-dependent processes in exocytosis.

The finding that brain membrane EGTA extract alone is able

• to replace cytosol in supporting Ca2+-triggered NE secretion

 in PC12 cells is somewhat surprising. We suggest that the likely explanation is 2-fold.

1. some cytosolic proteins essential for exocytosis have a membrane-bound pool
   within permeabilized cells, whose activity might be sufficient for a normal level of exocytosis.
2. although the 100,000 3 g membrane pellet was washed to remove as many cytosolic proteins as possible,

• some cytosolic proteins that associate with membranes in a
  • Ca2+-independent manner are probably present in the membrane EGTA extract.
• these proteins likely constitute only a small percentage of the proteins in the extract, as
  • the characteristics of the activity triggered by the membrane extract
  • are quite different to that of cytosol (Fig. 2).

 Using an unbiased biochemical purification method, we demonstrated that

•  calmodulin and PKC directly modulate the exocytotic machinery downstream of Ca2+ entry
• they signal through membrane-attached molecules to increase exocytosis.

 These targets include integral and peripheral membrane proteins, and cytosolic proteins that have a significant
membrane-bound pool.  The modest stimulation by calmodulin and PKC on secretion might suggest a regulatory

role. However, it is also possible that some intermediates in their signaling pathways are in limiting amounts in the
cell ghosts, so that their full effects were not observed. Half-maximal stimulation was obtained at

• about 3 nM for PKC and
• at about 75 nM for calmodulin.

This is consistent with an enzymatic role for PKC, and predicts a high-affinity interaction between

• calmodulin and its substrate protein.

 Ca2+ regulates exocytosis at many different levels. Prior studies indicated that Ca2+ signaling occurs in

• the priming steps as well
• as in triggering steps (49, 50).

Our priming triggering protocol 

1. does not allow Ca2+-dependent priming events to be assayed, as EGTA is present in the priming reaction.
2. a different approach revealed the existence of both high and low Ca2+-dependent processes (Fig. 2).
3. this analysis indicated that late triggering events require high [Ca2+], whereas
4. early priming events require low [Ca2+]. If, as proposed, there is

a pronounced intracellular spatial and temporal [Ca2+] gradient from

• the point of Ca2+ entry during depolarization (51),
• perhaps triggered events occur closer to the point of Ca2+ entry,
• while Ca2+-dependent priming events occur further away from the point of Ca2+ entry.

Distinct Ca2+ sensors at these stages might be appropriately tuned to different [Ca2+] to handle different tasks.

By analyzing the Ca2+ sensitivity of calmodulin-and PKC-stimulated release, we addressed the question of

• whether calmodulin and PKC plays an early or a late role in vesicle release.
•  they both require relatively high [Ca2+] (Fig. 8B),
• implying that calmodulin and PKC both mediate late triggering events, consistent with some earlier reports
  (34, 52, 53).

In addition, it is interesting to note that PKC does not alter the calcium sensitivity of release in cracked cells, in contrast

to observations from the chick ciliary ganglion (36). Therefore, in contrast to previous electrophysiological studies (28),
we are able to limit the possible modes of PKC action in our system to an increase in the readily releasable vesicle pool or
release sites, or an enhancement of the probability of release of individual vesicles upon Ca2+ influx.

The experiments assaying the calcium sensitivity of release (Figs. 2, 5, and 8) demonstrated

• a drop in release at very high [Ca2+].

This decline in release at high [Ca2+] has been previously reported (49, 51), and may represent

• the true Ca2+ sensitivity of the Ca2+-sensing mechanism inside cells.

However, in our system, it could also be due to the activation of a variety of Ca2+ -activated proteases, as experiments are usually performed in the presence of crude extracts, which include unsequestered proteases.

What might the molecular targets of PKC and calmodulin be? An obvious calmodulin target molecule is CaMKII.

• but calmodulin’s effect on exocytosis is ATP-independent, rendering the involvement of a kinase unlikely.

 Calmodulin has also been shown to associate with

• synaptic vesicles in a Ca2+-dependent fashion through synaptotagmin (54),
• probably by binding to its C-terminal tail (55), and to promote Rab3A dissociation from synaptic vesicles (56).
• However, there was little calcium-dependent binding of calmodulin to synaptotagmin
  • either on synaptic vesicles, in a bead binding assay with recombinant proteins,
  • or in a calmodulin overlay (data not shown).

In addition, using immobilized calmodulin, we did not see

• significant Ca2+-dependent pull-down of synaptotagmin or Rab3A from rat brain extract (data not shown).

Recent work has suggested three other candidate targets for calmodulin, Munc13, Pollux, and CRAG (57).

• Pollux has similarity to a portion of a yeast Rab GTPase-activating protein, while
• CRAG is related to Rab3 GTPase exchange proteins.

Further work is required to investigate the role of their interactions with calmodulin in vivo.

The recent report that calmodulin mediates yeast vacuole fusion (24) is intriguing, as it raises the possibility that

• calmodulin, a highly conserved ubiquitous molecule,
  • may mediate many membrane trafficking events.

It is not yet known if

• the effector molecule of calmodulin is conserved or variable across species and different trafficking steps.

It is enticing to propose a model for Ca2+ sensing whereby

• calmodulin is a high affinity Ca2+ sensor for both constitutive and regulated membrane fusion.

1. In the case of constitutive fusion, calmodulin may be the predominant Ca2+ sensor.
2. In the case of slow, non-local exocytosis of large dense core granules, an additional requirement for
3. the concerted actions of other molecule(s) that are better tuned to intermediate rises in [Ca2+] might exist.

At the highly localized sites of fast exocytosis of small clear vesicles where high [Ca2+] is reached,

• specialized low affinity sensor(s) are likely required
• in addition to calmodulin to achieve membrane fusion.

Therefore, although calmodulin participates in multiple types of vesicle fusion,

• the impact of Ca2+ sensing by calmodulin on vesicle release likely varies.

Due to the fact that calmodulin binding to some proteins can be modulated by PKC phosphorylation, one might suspect

• PKC action on exocytosis proceeds through a calmodulin-dependent pathway.
• but the effects of calmodulin and PKC are additive within our system,
  • suggesting that PKC does not act by releasing calmodulin from a substrate
    • that functions as a calmodulin storage protein.

How Ca2+ regulates presynaptic vesicle release has been an open question for many years. By

• identifying calmodulin and PKC as modulators of Ca21-regulated exocytosis and clarifying their functions,
• we have extended our knowledge of the release process.

While the basic machinery of membrane fusion is becoming better understood,

• the multiple effects of Ca2+ on exocytosis remain to be elucidated at the molecular level.

In addition, the ways that Ca2+ regulation may be important to

• the mechanisms of synaptic plasticity in the central nervous system

EXPERIMENTAL PROCEDURES

Materials
Rat Brain Cytosol Preparation
Membrane EGTA Extract Preparation

Cracked Cell Assay

PC12 cells were maintained and [3H]NE labeled as described previously (11). Labeled cells were harvested by pipetting with ice-cold potassium glutamate buffer (50 mM Hepes, pH 7.2, 105 mM potassium glutamate, 20 mM potassium acetate, 2 mM EGTA) containing 0.1% bovine serum albumin. Subsequent manipulations were carried out at 0–4 °C. Labeled cells (1–1.5 ml/dish) were mechanically permeabilized passage through a stainless steel homogenizer. The cracked cells were adjusted to 11 mM EGTA and

• incubated on ice for 0.5–3 h, followed by three washes in which
• the cells were centrifuged at 800 3 g for 5 min and
• resuspended in potassium glutamate buffer containing 0.1% bovine serum albumin.

Composite Assay 

Each release reaction contains 0.5–1 million cracked cells, 1.5 mM free Ca2+, 2 mM MgATP,
and the protein solution to be tested in potassium glutamate buffer. Release reactions were initiated
by incubation at 30 °C and terminated by returning to ice. The supernatant of each reaction was
isolated by centrifugation at 2,500 3 g for 30 min at 4 °C, and the

• released [3H]NE was quantified by scintillation counting (Beckman LS6000IC).

Cell pellets were dissolved in 1% Triton X-100, 0.02% azide and similarly counted. NE release

• was calculated as a percentage of total [3H] in the supernatant.

Priming Assay

A priming reaction contains about

• 1–2 million cracked cells,
• 2 mM MgATP, and
• the protein solution to be tested.
• Ca2+ is omitted.

The primed cells were spun down, washed once with fresh potassium glutamate buffer, and

• distributed into two triggering reactions, each containing
• rat brain cytosol and free Ca2+
• The triggering reaction was performed at 30 °C for 3 min, and
• the NE release was measured
  • as in a composite assay.

Triggering Assay

Cracked cells were primed …, centrifuged, washed …, and

distributed into triggering reactions containing

• 1.5 mM free Ca2+ and the protein solution 

To inhibit any ATP dependent activity in the triggering reaction,  an

• ATP depletion system of

1. 1. hexokinase
   2. MgCl2,
   3. glucose or

• a non-hydrolyzable ATP analogue AMPPNP

was added into the triggering reaction. NE release was measured as above.

Free Ca2+ Concentration Determination

The range of Ca2+free in the release reaction (Fig. 2B) was achieved

• by adding Ca2+ into potassium glutamate buffer to reach final [Ca2+] total values of
  • 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 1.9, and 2.0 mM.
• The pH of the reaction was 7.24 when no Ca2+ was added and
• 7.04 when 2.0 mM Ca2+ was added, 
  • in the absence of protein extracts or cracked cells.

Fig. 2B.   The range of [Ca21]free in the release reaction (Fig. 2B)

Free Ca2+ concentrations were determined using video microscopic
measurements of fura-2 fluorescence (41). [Ca2+]free was calculated from the equation

• [Ca2+]free 5 Kd*3 (R 2 Rmin)/(Rmax 2 R) (42).

The values of Rmin, Rmax, and Kd* were determined in the following solutions: 

potassium glutamate buffer (PGB) containing

• 8 x 3 10^6 cracked cells/ml, 2 mM MgATP (PGB+CC)

1) Rmin:  PGB+CC and 10 mM additional EGTA;

2) Rmax: PBG+CC, and 10 mM total Ca2+;

3) Kd*: PGB+CC, 28 mM additional EGTA, and 18 mM total Ca2+, pH 7.2
([Ca2+]free 5 = 169 nM, determined in the absence of cells and MgATP

• based on fura-2 calibration in cell-free solutions).

These solutions were

1. incubated at 37 °C ,
2. mixed with fura-2 pentapotassium salt
   (100 mM; Molecular Probes, Eugene, OR), and
3. imaged.

This procedure allowed us to take into account

• changes in fura-2 properties
• caused by the presence of
  • permeabilized cells.

Duplicate measurements of the above range of [Ca2+] total gave

• the following average [Ca2+] free values:
• 106, 146, 277, 462, 971, 1468, 1847, and 2484 nM.

Purification of Active Proteins

All procedures were carried out at 4 °C or on ice. Membrane
EGTA extract of one or two bovine brain(s) was

1. filtered through cheesecloth and
2. loaded overnight onto a column packed with DEAE-Sepharose
   CL-6B beads (Amersham Pharmacia Biotech).

The column was then

1. washed with
   (20 mM Hepes, pH 7.5, 0.25 mM sucrose, 2 mM EGTA, 1 mM dithiothreitol) and 
2. step eluted with 10 column volumes of elution buffer
   (20 mM Hepes, pH 7.5, 2 mM EGTA, 400 mM KCl, 1 mM dithiothreitol).
   100 ml of every other fraction was
3. dialyzed overnight into PGB, and
4. tested in a composite release assay for activity.

• The active fractions were pooled and dialyzed into zero salt buffer
  (20 mM Hepes, pH 7.5, 2 mM EGTA) and
• batch bound to 10 ml of Affi-Gel Blue beads (Bio-Rad) or DyeMatrex-Green A beads (Amicon)

Blue beads were used in earlier experiments, and Green beads were used later to

• specifically deplete CAPS, which was known to bind to Green beads (9).

The unbound material was

1. collected,
2. concentrated to about 2 ml using a Centriprep-10 (Amicon), and
3. loaded onto a 120-ml HiPrep Sephacryl S-200 gel filtration column
   (Amersham Pharmacia Biotech).

Samples were run on the S-200 column in PGB at a flow rate of 7 ml/h.

• 10–50 ml of every other fraction was tested for
  • activity in the cracked cell composite assay, and
• two peaks of activity were observed (Fig. 3).

The first peak of activity had a predicted molecular mass of 85 kDa.
The corresponding material was

• adjusted to 10 mM potassium phosphate concentration (pH 7.2) and
• loaded onto a 1-ml column packed with hydroxyapatite Bio-Gel HT
  (Bio-Rad).

The bound material was

• eluted with a linear K-PO4 gradient from 10 to 500 mM (pH 7.2)
•  at a flow rate of about 0.1 ml/min, and
• 0.4–0.5-ml fractions were collected.
•  each fraction was dialyzed into PGB and
• tested for activity.

The fractions were also analyzed by

• SDS-PAGE and silver staining (Sigma silver stain kit).

The active material was concentrated and resolved

• on an 8% poly-acrylamide gel.

Two Coomassie-stained protein bands that matched the activity profile (Fig. 6)

• were excised from the gel,
• sequenced by the Stanford PAN facility.

The two polypeptide sequences obtained from the upper band were:

1. LLNQEEGEYYNVPIXEGD
2. IRSTLNPRWDESFT.

The only bovine protein that contains both polypeptides is PKCa.
The four polypeptide sequences obtained from the lower band were:

1. YELTGKFERLIVGLMRPPAY,
2. LIEILASRTNEQIHQLVAA,
3. MLVVLLQGTREEDDVVSEDL, and
4. EMSGDVRDVFVAIVQSVK.

Based on these sequences, the protein band was

• unambiguously identified to be bovine annexin VI.

The second S-200 peak has a predicted molecular mass of 25 kDa.
The corresponding material was

• dialyzed into zero salt buffer
  (20 mM Tris, pH 7.5, 1 mM EGTA) and
• injected onto a Mono-Q HR 5/5 FPLC column
  (Pharmacia).

The FPLC run was performed at 18 °C at 1 ml/min and

• 1-ml fractions were collected
• with a linear salt gradient from 0 to 1 M KCl over 71 ml.

The fractions containing proteins (determined by A280) were

• dialyzed into PGB and
• tested in the cracked cell assay.

Western Blot

Anti-calmodulin antibody and anti-PKC antibody were used, and

• ECL (Amersham) was used for detection.

RESULTS

A Membrane EGTA Extract Supports NE Release 

Brain cytosol, prepared as the supernatant of the brain homogenate,

• effectively stimulates NE release
• in the cracked cell assay (Fig. 1)
  as previously shown (9). 

We wondered whether crude extracts other than cytosol

• could support NE release, and we focused on
• extractable peripheral membrane proteins.

We found that a salt or EGTA extract of brain membranes,
membranes defined as the

• 100,000 3 g pellet of the crude homogenate,
• reconstituted secretion in the absence of cytosol.
• the salt extract only slightly enhanced NE release
  above background (data not shown), the 

EGTA extract not only stimulated NE release to a high level,

• similar to that supported by cytosol, but also
• had a higher specific activity than cytosol (Fig. 1). 

FIG. 1. The EGTA extract of brain membranes can support NE release in the absence of cytosol. Rat brain membrane EGTA extract (closed triangles) and rat brain cytosol (closed squares) were prepared as described under “Experimental Procedures.” NE release was measured in a composite reaction mixture of cracked cells, MgATP, Ca2+, and the indicated amount of crude extracts.

The ability of the membrane EGTA extract to support secretion is consistent with the fact that

• following cracking, the cells are immediately extracted with EGTA, and are presumably
• devoid of most membrane EGTA-extractable factors.

This also suggests that these factors, some of which are probably

• Ca2+-dependent membrane-associating proteins,
• participate in Ca2+- triggered exocytosis.

The Membrane EGTA Extract Is Enriched in Triggering Fators

NE release in cracked cells can be resolved into two sequential stages,

• an ATP-dependent priming stage and
• an ATP-independent Ca21-dependent triggering stage (11), and
• proteins can be tested for activity in either stage.

An effect in priming indicates

1. an early role for the protein, and
2. an effect in triggering a late ATP-independent role.

Since the protein composition of the

• membrane EGTA extract and cytosol are different,

we tested whether they had different activities

• in the priming stage versus the triggering stage.

We found that the membrane EGTA extract is enriched in factors that

• act during triggering stage of NE release, as
• the same amount of protein from the membrane EGTA extract as cytosol
• gave a higher stimulation in the triggering assay, but
• not in the priming assay (Fig. 2A). 

Regular cytosol is prepared in a buffer containing 2 mM EGTA, and thus

• presumably contains some of the proteins present in the membrane EGTA extract.

Cytosol prepared in the absence of EGTA showed an even lower specific activity

• in the triggering assay compared with regular cytosol (Fig. 2A).

Identification of Calmodulin as an Active Triggering Factor in the EGTA Extract

Biochemical fractionation of the bovine brain membrane EGTA extract was carried out

• to identify the active components capable of reconstituting NE release.

Activity was assayed in a composite reaction mixture containing

• cracked cells,
• ATP,
• Ca2+, and
• the test protein(s).

Except for the presence of bovine serum albumin in the basal buffer,

• no other proteins were added to the cell ghosts except for the test protein(s).

Initial tests indicated that at least

1. part of the activity in the membrane EGTA extract binds to and
2. can be efficiently eluted from an anion exchanger and hydroxyapatite resin,
3. but does not bind to Amicon color resins.

The starting material was, therefore, sequentially purified using

• DEAE, Affi-Gel Blue (or Matrex Green-A), and gel filtration chromotography.

Gel filtration fractionation indicated the presence of two peaks of activity with

• predicted molecular masses of 25 and 85 kDa, respectively (Fig. 3).

FIG. 3. Gel filtration chromatography reveals two stimulatory factors in the membrane EGTA extract.

In order to purify the active component(s) in the membrane EGTA extract, the crude extract from one bovine brain was fractionated chromatographically (see Experimental Procedures” for details). Fractions from a Sephacryl S-200 gel filtration column were tested for their activity in stimulating NE release in the composite assay. The two activity peaks have predicted molecular masses of 85 and 25 kDa, respectively. The arrows indicate the retention volume of standard proteins run on the same column.

The low molecular weight active factor was purified to homogeneity, as judged by a

• Coomassie-stained SDS-PAGE gel, after a subsequent Mono-Q fractionation (Fig. 4).

FIG. 4. The low molecular wen.ight active factor is calmodulin

A, the  membrane EGTA extract from one bovine brain (Start) was subjected to sequential fractionation on DEAE, Blue A, and
Sephacryl S-200 columns. The pooled material containing the activity after each chromotographic step was analyzed by SDS-
PAGE and Coomassie staining. The arrowheads indicate the presence of calmodulin in all the lanes. Calmodulin shows a
mobility shift depending on whether or not Ca2+ is present during electrophoresis (see panel C).
B, the active material  pooled from Sephacryl S-200 was fractionated on a Mono-Q FPLC column and the fractions
(5 ml/fraction) were tested for activity in a composite assay. The activity peak is shown.
C, the active Mono-Q fractions (5 ml/fraction) were subjected to SDS-PAGE in the presence of 1 mM EGTA or 0.1 mM Ca2+,
and the gels stained with Coomassie Blue.
D, fraction 47 (1 ml) was probed by Western blotting with a monoclonal anti-calmodulin antibody. No Ca2+ or EGTA was
added during SDS-PAGE.

We reasoned that the protein might be calmodulin (43) based on the following:

1) It is a relatively small protein (14–18 kDa) that is abundant in the
starting extract (Fig. 4A).
2) It elutes at a very high salt concentration (0.41 M KCl) on the
Mono-Q column.
3) It stains negatively in silver stain (data not shown).
4) Its electrophoretic mobility shifts depending on the presence or
absence of Ca21 (Fig. 4C).

A Western blot with an anti-calmodulin monoclonal antibody gave a
positive signal (Fig. 4D), confirming our prediction.

Properties of Calmodulin-stimulated Exocytosis

We used commercial calmodulin or bacterially expressed recombinant calmodulin to confirm our purification result; both sources of authentic calmodulin stimulated NE release as expected. Moreover, we found that calmodulin stimulates secretion in a triggering assay as well as in a composite assay (Fig. 5A).

The half-maximal increase was at 75 nM (250 ng/200 ml) final calmodulin concentration. This is within the broad
range of affinities between calmodulin and its various targets and suggests that the interaction between
calmodulin and its target molecule in exocytosis is in the physiological range. When the triggering reaction was
performed at different Ca2+ concentrations, calmodulin increased NE release only at high [Ca2+] (0.4 – 2 mM)
similar to the crude EGTA extract (Fig. 5B),

suggesting that calmodulin contributes to the triggering activity of the membrane EGTA extract.  Calmodulin’s affinity for Ca2+ has
been  reported to be around 1 mM (25),

• consistent with the Ca2+ requirement for
• calmodulin-stimulated secretion that we observed.

FIG. 5. Calmodulin stimulates NE release in the triggering stage.
A, calmodulin (obtained from Sigma) increased NE release in the
triggering assay in a dose-dependent fashion, in the absence of ATP
or any other cytosolic proteins. In this particular experiment, the
maximal release achieved by addition of rat brain cytosol was 46.5%.

B, the triggering assay was performed with different concentrations
of free Ca2+. Calmodulin (3 mg bacterially expressed recombinant
protein; closed squares) increased NE release with a similar Ca2+
sensitivity to rat brain membrane EGTA extract (10 mg; closed
triangles), as compared with conditions in which no protein was
added (open squares).

Western analysis with commercial protein as standards indicated that calmodulin 

•  constitutes about 5% of total proteins in the rat brain membrane EGTA extract
• and about 2% of total proteins in the rat brain cytosol (data not shown).

In addition, a significant amount of calmodulin appears to be left

• in the washed cell ghosts (data not shown).

Based on the activity of saturating levels of

• pure calmodulin (releasing 6–10% of total [3H]NE)
• and crude EGTA extract (releasing ;45% of total [3H]NE),

we estimated that

• calmodulin accounts for 13–22% of total activity of the extract.

Consistent with this,

• a high affinity calmodulin-binding peptide
  (CaMKIIa(291–312) (44), used at 5 mM) and
• an anti-calmodulin antibody (2 mg/200 ml)
• inhibited about 20% of the membrane EGTA extract-stimulated release
  (6.7 mg of extract added; data not shown).

We showed that calmodulin increased NE release

• in the triggering stage.

Since regular triggering reactions were performed

• in the absence of any added ATP,

this suggests that

• calmodulin enhanced secretion in an ATP-independent fashion.

Furthermore, residual ATP in the cell ghosts did not play a role, since

•  addition of a hexokinase ATP depletion system that
• can deplete millimolar concentrations of ATP
  • within a few minutes (11) had little effect, as did
  • addition of 5 mM AMPPNP,
• which blocks ATP-dependent enzymatic activity (Fig.8A).

Therefore, we ruled out the possibility that a kinase mediates calmodulin’s effect.

FIG. 8. PKC and calmodulin stimulate the late triggering reaction in
an ATP-dependent and ATP-independent manner respectively.
A, triggering assays were performed to test the activity of calmodulin
(recombinant; black bars) and PKC (purified rat brain PKC from
Calbiochem; shaded bars) in the absence of ATP. A regular triggering
assay is done in the absence of ATP (2ATP). To deplete residual ATP
in the cells, hexokinase-based ATP depletion was employed (1Hexo).
Alternatively, 5 mM AMP-PNP (1AMP-PNP) was added in the triggering
reaction. Under all three conditions, calmodulin increased release
as compared with the background (buffer only; white bars), whereas
PKC did not.
B, NE release in a composite assay was measured with varying
concentrations of free Ca2+ in the presence of 10 mg of calmodulin
(recombinant; closed triangles), 70 ng of PKC  (purified rat brain PKC
from Calbiochem; closed squares), or buffer only (open squares).

A series of calmodulin mutants from Paramecium and chicken were tested

• for their ability to enhance Ca2+-stimulated secretion, and
• none of the mutations abolished the calmodulin effect (data not shown).

These mutations include

• S101F, M145V, E54K, G40E/D50N, V35I/D50N within Paramecium
• calmodulin (45), and M124Q, M51A/V55A, and M51A/V55A/L32A
  within chicken calmodulin (46, 47).

The Paramecium calmodulin mutants are the result of

• naturally occurring mutations that result in aberrations in their behavior.

These mutants can be grouped into two categories according to their
behavior, reflecting their loss of either

1. a Ca2+-dependent Na1 current
    (calmodulin N-terminal lobe mutants: E54K, G40E/D50N, and
    V35I/D50N) or
2. a Ca21-dependent K1 current
   (calmodulin C-terminal lobe mutants: S101F and M145V) (45).

The chicken calmodulin mutants have been shown to

• differentially activate myosin light chain kinase
  (M124Q, M51A/V55A, and M51A/V55A/L32A),
  CaMKII (M124Q),  and CaMKIV (M124Q),

and the mutated residues are thought to be important in

• defining calmodulin’s binding specificity (46, 47).

Our finding that these mutant calmodulins can stimulate exocytosis suggests that

• calmodulin-binding domains similar to those of Paramecium Ca2+/calmodulin-dependent
  ion channels, myosin light chain kinase, CaMKII, and CaMKIV,
• are unlikely to mediate release utilizing the conserved SNARE fusion machinery, as they
• could be completely abolished by addition of exogenous syntaxin H3 domains (data not shown).
• the same molecular pathway was not activated, since their effects were additive (data not shown).

 

Acknowledgments—

We thank Diana Bautista and Dr. Richard S.Lewis for generous help

with [Ca21]free determination; Dr. Ching Kung for providing the Paramecium calmodulin

mutants, and Dr. Anthony R. Means for providing the chicken calmodulin mutants. We also

thank Dr. Jesse C. Hay for the initial setup of the cracked cell assay, and Dr. Suzie J.

Scales for helpful comments on the manuscript.

REFERENCES

1. Calakos, N., and Scheller, R. H. (1996) Physiol. Rev. 76, 1–29

2. Su¨ dhof, T. C. (1995) Nature 375, 645–653

3. Zucker, R. S. (1996) Neuron 17, 1049–1055

4. Hanson, P. I., Heuser, J. E., and Jahn, R. (1997) Curr. Opin. Neurobiol. 7, 310–315

5. Chen, Y. A., Scales, S. J., Patel, S. M., Doung, Y.-C., and Scheller, R. H. (1999) Cell 97, 165–174

6. Neher, E., and Zucker, R. S. (1993) Neuron 10, 21–30

7. Kamiya, H., and Zucker, R. S. (1994) Nature 371, 603–606

SOURCE

J Biol Chem 1999; 274(37): 25469-26476.

Other related articles published in this Open Access Online Scientific Journal include the following:

The role of ion channels in Na(+)-K(+)-ATPase: regulation of ion transport across the plasma membrane has been studies by our Team in 2012 and 2013. 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

http://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

http://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

http://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

http://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

• Atrioventricular (AV) Conduction Disease (block): Human Mutations affecting the Voltage Clock

Aviva Lev-Ari, PhD, RN

• Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis

Aviva Lev-Ari, PhD, RN

•  Genomics & Genetics of Cardiovascular Disease Diagnoses: A Literature Survey of AHA’s Circulation Cardiovascular Genetics, 3/2010 – 3/2013

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

• Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

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

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

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

http://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

This study presents  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

Other related articles published on this Open Access Online Scientific Journal include the following:

ION CHANNEL and Cardiovascular Diseases

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

Calcium Role in Cardiovascular Diseases

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton
Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-
that-are-related-to-the-actin-cytoskeleton/

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-
skeleton-and-lipid-structures-in-signaling-and-cell-motility/

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease
Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-
exchange-mechanism-in-health-and-disease/

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and
Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia,
Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-
involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-
post-ischemic-arrhythmia-similarities-and-differen/

Part V: Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

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

http://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocytosis/

Part VI: 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
http://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/

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure –
Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-
and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells:
The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-
muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction
(Ryanopathy) and Calcium as Neurotransmitter Sensor
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-
and-calcium-release-related-contractile-dysfunction-ryanopathy/

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

Part XI: Sensors and Signaling in Oxidative Stress
Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

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

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

http://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

 

Mitochondria and its Role in Cardiovascular Diseases

Mitochondria and Oxidative Stress Role in Cardiovascular Diseases Reversal of Cardiac Mitochondrial Dysfunction
Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

Calcium Signaling, Cardiac Mitochondria and Metabolic Syndrome
Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/11/09/calcium-signaling-cardiac-mitochondria-and-metabolic-syndrome/

Mitochondrial Dysfunction and Cardiac Disorders
Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-dysfunction-and-cardiac-disorders/

Mitochondrial Metabolism and Cardiac Function
Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing
Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2013/04/14/chapter-5-mitochondria-and-cardiovascular-disease/

MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix Identified
Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/02/03/mit-scientists-on-proteomics-all-the-proteins-
in-the-mitochondrial-matrix-identified/

Mitochondrial Dynamics and Cardiovascular Diseases
Ritu Saxena, Ph.D.
http://pharmaceuticalintelligence.com/2012/11/14/mitochondrial-dynamics-and-cardiovascular-diseases/

Mitochondrial Damage and Repair under Oxidative Stress
Larry H Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Nitric Oxide has a Ubiquitous Role in the Regulation of Glycolysis -with a Concomitant Influence on Mitochondrial Function
Larry H. Bernstein, MD, FACP
http://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-
glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Mitochondrial Mechanisms of Disease in Diabetes Mellitus
Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2012/08/01/mitochondrial-mechanisms-of-disease-in-diabetes-mellitus/

Mitochondria Dysfunction and Cardiovascular Disease – Mitochondria: More than just the “Powerhouse of the Cell”
Ritu Saxena, PhD
http://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

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

Posted in Biological Networks, Gene Regulation and Evolution, Biomedical Measurement Science, Calcium Signaling, Cell Biology, Signaling & Cell Circuits, tagged Aviva Lev-Ari, Bernard Katz, Heart Failure, Howard Hughes Medical Institute, James Rothman, Justin Pearlman, Larry H. Bernstein, Richard Scheller, SNARE, Stanford University School of Medicine, Thomas C. Südhof, Vascular smooth muscle on September 10, 2013| 11 Comments »

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

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

Image created by Adina Hazan 06/30/2021

This article is the Part X in a series of articles on Activation and Dysfunction of the Calcium Release Mechanisms in Cardiomyocytes and Vascular Smooth Muscle Cells.

The Series consists of the following articles:

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton

Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

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

http://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/

Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

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

http://pharmaceuticalintelligence.com/2013/09/02/renal-distal-tubular-ca2-exchange-mechanism-in-health-and-disease/

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

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

http://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-differen/

Part V: Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

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

http://pharmaceuticalintelligence.com/2013/12/23/calmodulin-and-protein-kinase-c-drive-the-ca2-regulation-of-hormone-and-neurotransmitter-release-that-triggers-ca2-stimulated-exocytosis/

Part VI: 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

http://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/

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmiasand Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

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

http://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

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

http://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor

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

http://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-and-calcium-release-related-contractile-dysfunction-ryanopathy/

Part X: 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

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

Part XI: Sensors and Signaling in Oxidative Stress

Larry H. Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2013/11/01/sensors-and-signaling-in-oxidative-stress/

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

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

http://pharmaceuticalintelligence.com/2013/12/21/genetic-polymorphisms-of-ion-channels-have-a-role-in-the-pathogenesis-of-coronary-microvascular-dysfunction-and-ischemic-heart-disease/

Introduction

Author: Larry H Bernstein, MD, FCAP 

This introduction is based on two sources:

#1:

Michael J. Berridge, Smooth muscle cell calcium activation mechanisms

The Babraham Institute, Babraham, Cambridge CB22 4AT, UK

J Physiol 586.21 (2008) pp 5047–5061

http://jp.physoc.org/content/586/21/5047.full.pdf

and

#2

Thomas C Südhof, A molecular machine for neurotransmitter release: synaptotagmin and beyond

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

Part IX of this series of articles discussed the mechanism of the signaling of smooth muscle cells by the interacting parasympathetic neural innervation that occurs by calcium triggering neurotransmitter release by initiating synaptic vesicle fusion.   It involves the interaction of soluble N-acetylmaleimide-sensitive factor (SNARE) and SM proteins, and in addition, the discovery of a calcium-dependsent Syt1 (C) domain of protein- kinase C isoenzyme, which binds to phospholipids.  It is reasonable to consider that it differs from motor neuron activation of skeletal muscles, mainly because the innervation is in the involuntary domain.   The cranial nerve rooted innervation has evolved comes from the spinal ganglia at the corresponding level of the spinal cord.  It is in this specific neural function that we find a mechanistic interaction with adrenergic hormonal function, a concept intimated by the late Richard Bing.  Only recently has there been a plausible concept that brings this into serious consideration.  Moreover, the review of therapeutic drugs that are used in blocking adrenergic receptors are closely related to the calcium-channels.  Interesting too is the participation of a phospholipid bound protein-kinase isoenzyme C calcium-dependent domain Syt1.  The neurohormonal connection lies in the observation by Katz in the 1950’s that the vesicles of the neurons hold and eject fixed amounts of neurotransmitters.

In Sudhof’s Lasker Award presentation he refers to the biochemical properties of synaptotagmin were found to precisely correspond to the extraordinary calcium-triggering properties of release, and to account for a regulatory pathway that also applies to other types of calcium-triggered fusion, for example fusion observed in hormone secretion and fertilization. At the synapse, finally, these interdependent machines — the fusion apparatus and its synaptotagmin-dependent control mechanism — are embedded in a proteinaceous active zone that links them to calcium channels, and regulates the docking and priming of synaptic vesicles for subsequent calcium-triggered fusion. Thus, work on neurotransmitter release revealed a hierarchy of molecular machines that mediate the fusion of synaptic vesicles, the calcium-control of this fusion, and the embedding of calcium-controlled fusion in the context of the presynaptic terminal at the synapse.  The neural transmission is described as a biological relay system. Neurotransmission kicks off with an electrical pulse that runs down a nerve cell, or neuron. When that signal reaches the tip, calcium enters the cell. In response, the neuron liberates chemical messengers—neurotransmitters—which travel to the next neuron and thus pass the baton.

He further stipulates that synaptic vesicle exocytosis operates by a general mechanism of membrane fusion that revealed itself to be a model for all membrane fusion, but that is uniquely regulated by a calcium-sensor protein called synaptotagmin.  Neurotransmission is thus a combination of electrical signal and chemical transport.

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

Several SMC types illustrate how signaling mechanisms have been adapted to control different contractile functions with particular emphasis on how Ca2+ signals are activated.

[1] Neural activation of vas deferens smooth muscle cells

Noradrenaline (NA) acts by stimulating α1-adrenoreceptors to produce InsP3, which then releases Ca2+ that may induce an intracellular Ca2+ wave similar to that triggered by the ATP-dependent entry of external Ca2+. In addition, the α1-adrenoreceptors also activate the smooth muscle Rho/Rho kinase signalling pathway that serves to increase the Ca2+ sensitivity of the contractile machinery.

[2] Detrusor smooth muscle cells

The bladder, which functions to store and expel urine, is surrounded by layers of detrusor SMCs. The latter have two operational modes: during bladder filling they remain relaxed but contract vigorously to expel urine during micturition. The switch from relaxation to contraction, which is triggered by neurotransmitters released from parasympathetic nerves, depends on the acceleration of an endogenous membrane oscillator that produces the repetitive trains of action potentials that drive contraction.

This mechanism of activation is also shared by [1], and uterine contraction.  SMCs are activated by membrane depolarization (ΔV) that opens L-type voltage-operated channels (VOCs) allowing external Ca2+ to flood into the cell to trigger contraction. This depolarization is induced either by ionotropic receptors (vas deferens) or a membrane oscillator (bladder and uterus). The membrane oscillator, which resides in the plasma membrane, generates the periodic pacemaker depolarizations responsible for the action potentials that drive contraction.

The main components of the membrane oscillator are the Ca2+ and K+ channels that sequentially depolarize and hyperpolarize the membrane, respectively. This oscillator generates the periodic pacemaker depolarizations that trigger each action potential. The resulting Ca2+ signal lags behind the action potential because it spreads into the cell as a slower Ca2+ wave mediated by the type 2 RYRs.

Neurotransmitters such as ATP and acetylcholine (ACh), which are released from parasympathetic axonal varicosities that innervate the bladder, activate or accelerate the oscillator by inducing membrane depolarization (ΔV).

[3]  The depolarizing signal that activates gastrointestinal, urethral and ureter SMCs is as follows:

A number of SMCs are activated by pacemaker cells such as the interstitial cells of Cajal (ICCs) (gastrointestinal and urethral SMCs) or atypical SMCs (ureter). These pacemaker cells have a cytosolic oscillator that generates the repetitive Ca2+ transients that activate inward currents that spread through the gap junctions to provide the depolarizing signal (ΔV) that triggers contraction.

[4]  Our greatest interest has been in this mechanism.  The rhythmical contractions of vascular, lymphatic, airway and corpus cavernosum SMCs depend on an endogenous pacemaker driven by a cytosolic Ca2+ oscillator that is responsible for the periodic release of Ca2+ from the endoplasmic reticulum. The periodic pulses of Ca2+ often cause membrane depolarization, but this is not part of the primary activation mechanism but has a secondary role to synchronize and amplify the oscillatory mechanism. Neurotransmitters and hormones act by modulating the frequency of the cytosolic oscillator.

Vascular or airway SMCs are driven by a cytosolic oscillator that generates a periodic release of Ca2+ from the endoplasmic reticulum that usually appears as a propagating Ca2+ wave.

Step 1. The initiation and/or modulation of this oscillator depends upon the action of transmitters and hormones such as ACh, 5-HT, NA and endothelin-1 (ET-1) that increase the formation of InsP3 and diacylglycerol (DAG), both of which promote oscillatory activity.

Step 2. The oscillator is very dependent on Ca2+ entry to provide the Ca2+ necessary to charge up the stores for each oscillatory cycle. The nature of these entry mechanisms vary between cell types.

Step 3. The entry of external Ca2+ charges up the ER to sensitize the RYRs and InsP3 receptors prior to the next phase of release. An important determinant of this sensitivity is the luminal concentration of Ca2+ and as this builds up the release channels become sensitive to Ca2+ and can participate in the process of Ca2+-induced Ca2+ release (CICR), which is responsible for orchestrating the regenerative release of Ca2+ from the ER. The proposed role of cyclic ADP-ribose (cADPR) in airway SMCs is consistent with this aspect of the model on the basis of its proposed action of stimulating the SERCA pump to enhance store loading and such a mechanism has been described in colonic SMCs.

Step 4. The mechanism responsible for initiating Ca2+ release may depend either on the RYRs or the InsP3 receptors (I). RYR channels are sensitive to store loading and the InsP3 receptors will be sensitized by the agonist-dependent formation of InsP3.

Step 5. This initial release of Ca2+ is then amplified by regenerative Ca2+ release by either the RYRs or InsP3 receptors, depending on the cell type.

Step 6. The global Ca2+ signal then activates contraction.

Step 7. The recovery phase depends on the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), that pumps some of the Ca2+ back into the ER, and the plasma membrane Ca2+-ATPase (PMCA), that pumps Ca2+ out of the cell.

Step 8. One of the effects of the released Ca2+ is to stimulate Ca2+-sensitive K+ channels such as the BK and SK channels that will lead to membrane hyperpolarization. The BK channels are activated by Ca2+ sparks resulting from the opening of RYRs.

Step 9.  Another action of Ca2+ is to stimulate Ca2+-sensitive chloride channels (CLCA) (Liu & Farley, 1996; Haddock & Hill, 2002), which result in membrane depolarization to activate the CaV1.2 channels that introduce Ca2+ into the cell resulting in further membrane depolarization (ΔV).

Step 10. This depolarization can spread to neighbouring cells by current flow through the gap junctions to provide a synchronization mechanism in those cases where the oscillators are coupled together to provide vasomotion.

SOURCE

Smooth muscle cell calcium activation mechanisms. Berridge MJ.
J Physiol. 2008; 586(Pt 21):5047-61.   http://dx.doi.org/10.1113/jphysiol.2008.160440

Synaptotagmin functions as a Calcium Sensor

Thomas C. Südhof is at the Department of Molecular and Cellular Physiology and the Howard Hughes Medical Institute, Stanford University School of Medicine, Palo Alto, California, USA

Prof.  Thomas C. Südhof explains:

Fifty years ago, Bernard Katz’s seminal work revealed that calcium triggers neurotransmitter release by stimulating ultrafast synaptic vesicle fusion. But how a presynaptic terminal achieves the speed and precision of calcium-triggered fusion remained unknown. My colleagues and I set out to study this fundamental problem more than two decades ago.

How do the synaptic vesicle and the plasma membrane fuse during transmitter release? How does calcium trigger synaptic vesicle fusion? How is calcium influx localized to release sites in order to enable the fast coupling of an action potential to transmitter release? Together with contributions made by other scientists, most prominently James Rothman, Reinhard Jahn and Richard Scheller, and assisted by luck and good fortune, we have addressed these questions over the last decades.

As he described below, we now know of a general mechanism of membrane fusion that operates by the interaction of SNAREs (for soluble N-ethylmaleimide–sensitive factor (NSF)-attachment protein receptors) and SM proteins (for Sec1/Munc18-like proteins). We also have now a general mechanism of calcium-triggered fusion that operates by calcium binding to synaptotagmins, plus a general mechanism of vesicle positioning adjacent to calcium channels, which involves the interaction of the so-called RIM proteins with these channels and synaptic vesicles. Thus, a molecular framework that accounts for the astounding speed and precision of neurotransmitter release has emerged. In describing this framework, I have been asked to describe primarily my own work. I apologize for the many omissions of citations to work of others; please consult a recent review for additional references1.

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

Outlook

Our work, together with that of other researchers, uncovered a plausible mechanism explaining how membranes undergo rapid fusion during transmitter release, how such fusion is regulated by calcium and how the calcium-controlled fusion of synaptic vesicles is spatially organized in the presynaptic terminal. Nevertheless, many new questions now arise that are not just details but of great importance. For example, what are the precise physicochemical mechanisms underlying fusion, and what is the role of the fusion mechanism we outlined in brain diseases? Much remains to be done in this field.

How calcium controls membrane fusion

The above discussion describes the major progress that was made in determining the mechanism of membrane fusion. At the same time, my laboratory was focusing on a question crucial for neuronal function: how is this process triggered in microseconds when calcium enters the presynaptic terminal?

While examining the fusion machinery, we wondered how it could possibly be controlled so tightly by calcium. Starting with the description of synaptotagmin-1 (Syt1)5, we worked over two decades to show that calcium-dependent exocytosis is mediated by synaptotagmins as calcium sensors.

Synaptotagmins are evolutionarily conserved transmembrane proteins with two cytoplasmic C2 domains (Fig. 3a)5,6. When we cloned Syt1, nothing was known about C2 domains except that they represented the ‘second constant sequence’ in protein-kinase C isozymes. Because protein kinase C had been shown to interact with phospholipids by an unknown mechanism, we speculated that Syt1 C2 domains may bind phospholipids, which we indeed found to be the case5. We also found that this interaction is calcium dependent6,7 and that a single C2 domain mediates calcium-dependent phospholipid binding (Fig. 3b)8. In addition, the Syt1 C2 domains also bind syntaxin-1 and the SNARE complex6,9. All of these observations were first made for Syt1 C2 domains, but they have since been generalized to other C2 domains.

As calcium-binding modules, C2 domains were unlike any other calcium-binding protein known at the time. Beginning in 1995, we obtained atomic structures of calcium-free and calcium-bound Syt1 C2 domains10 in collaboration with structural biologists, primarily Jose Rizo (Fig. 3c). These structures provided the first insights into how C2 domains bind calcium and allowed us to test the role of Syt1 calcium binding in transmitter release11.

The biochemical properties of Syt1 suggested that it constituted Katz’s long-sought calcium sensor for neurotransmitter release. Initial experiments in C. elegans and Drosophila, however, disappointingly indicated otherwise. The ‘synaptotagmin calcium-sensor hypothesis’ seemed unlikely until our electrophysiological analyses of Syt1 knockout mice revealed that Syt1 is required for all fast synchronous synaptic fusion in forebrain neurons but is dispensable for other types of fusion (Fig. 4)12. These experiments established that Syt1 is essential for fast calcium-triggered release, but not for fusion as such.

Although the Syt1 knockout analysis supported the synaptotagmin calcium-sensor hypothesis, it did not exclude the possibility that Syt1 positions vesicles next to voltage-gated calcium channels (a function now known to be mediated by RIMs and RIM-BPs; see below),

with calcium binding to Syt1 performing a role unrelated to calcium sensing and transmitter release. To directly test whether calcium binding to Syt1 triggers release, we introduced a point mutation into the endogenous mouse Syt1 gene locus. This mutation decreased the Syt1 calcium-binding affinity by about twofold11. Electrophysiological recordings revealed that this mutation also decreased the calcium affinity of neurotransmitter release approximately twofold, formally proving that Syt1 is the calcium sensor for release (Fig. 5). In addition to mediating calcium triggering of release, Syt1 controls (‘clamps’) the rate of spontaneous release occurring in the absence of action potentials, thus serving as an essential mediator of the speed and precision of release by association with SNARE complexes and phospholipids (Fig. 6a,b).

It was initially surprising that the Syt1 knockout produced a marked phenotype because the brain expresses multiple synaptotagmins6. However, we found that only three synaptotagmins—Syt1, Syt2 and Syt9—mediate fast synaptic vesicle exocytosis13. Syt2 triggers release faster, and Syt9 slower, than Syt1. Most forebrain neurons express only Syt1, but not Syt2 or Syt9, accounting for the profound Syt1 knockout phenotype. Syt2 is the predominant calcium sensor of very fast synapses in the brainstem14, whereas Syt9 is primarily present in the limbic system13. Thus, the kinetic properties of Syt1, Syt2 and Syt9 correspond to the functional needs of the synapses that contain them.

Parallel experiments in neuroendocrine cells revealed that, in addition to Syt1, Syt7 functions as a calcium sensor for hormone exocytosis. Moreover, experiments in olfactory neurons uncovered a role for Syt10 as a calcium sensor for insulin-like growth factor-1 exocytosis15, showing that, even in a single neuron, different synaptotagmins act as calcium sensors for distinct fusion reactions. Viewed together with results by other groups, these observations indicated that calcium-triggered exocytosis generally depends on synaptotagmin calcium sensors and that different synaptotagmins confer specificity onto exocytosis pathways.

We had originally identified complexin as a small protein bound to SNARE complexes (Fig. 6b)16. Analysis of complexin-deficient neurons showed that complexin represents a cofactor for synaptotagmin that functions both as a clamp and as an activator of calcium-triggered fusion17. Complexin-deficient neurons exhibit a phenotype milder than that of Syt1-deficient neurons, with a selective suppression of fast synchronous exocytosis and an increase in spontaneous exocytosis, which suggests that complexin and synaptotagmins are functionally interdependent.

How does a small molecule like complexin, composed of only ~130 amino acid residues, act to activate and clamp synaptic vesicles for synaptotagmin action? Atomic structures revealed that, when bound to assembled SNARE complexes, complexin contains two short a-helices flanked by flexible sequences (Fig. 6c). One of the a-helices is bound to the SNARE complex and is essential for all complexin function18. The second a-helix is required only for the clamping, and not for the activating function of complexin17. The flexible N-terminal sequence of complexin, conversely, mediates only the activating, but not the clamping, function of the protein. Our current model is that complexin binding to SNAREs activates the SNARE–SM protein complex and that at least part of complexin competes with synaptotagmin for SNARE complex binding. Calcium-activated synaptotagmin displaces this part of complexin, thereby triggering fusion-pore opening (Fig. 6a)1,18.

REFERENCES

1. Südhof, T.C. & Rothman, J.E. Membrane fusion: grappling with SNARE and SM proteins. Science 323, 474–477 (2009).

2. Hata, Y., Slaughter, C.A. & Südhof, T.C. Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin. Nature 366, 347–351 (1993).

3. Burré, J. et al. a-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science 329, 1663–1667 (2010).

4. Khvotchev, M. et al. Dual modes of Munc18–1/SNARE interactions are coupled by functionally critical binding to syntaxin-1 N-terminus. J. Neurosci. 27, 12147–12155 (2007).

5. Perin, M.S., Fried, V.A., Mignery, G.A., Jahn, R. & Südhof, T.C. Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature 345, 260–263 (1990).

6. Li, C. et al. Ca2+-dependent and Ca2+-independent activities of neural and nonneural synaptotagmins. Nature 375, 594–599 (1995).

7. Brose, N., Petrenko, A.G., Südhof, T.C. & Jahn, R. Synaptotagmin: a Ca2+ sensor on the synaptic vesicle surface. Science 256, 1021–1025 (1992).

8. Davletov, B.A. & Südhof, T.C. A single C2-domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid-binding. J. Biol. Chem. 268, 26386–26390 (1993).

9. Pang, Z.P., Shin, O.-H., Meyer, A.C., Rosenmund, C. & Südhof, T.C. A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent SNARE-complex binding in synaptic exocytosis. J. Neurosci. 26, 12556–12565 (2006).

10. Sutton, R.B., Davletov, B.A., Berghuis, A.M., Südhof, T.C. & Sprang, S.R. Structure of the first C2-domain of synaptotagmin I: a novel Ca2+/phospholipid binding fold. Cell 80, 929–938 (1995).

11. Fernández-Chacón, R. et al. Synaptotagmin I functions as a Ca2+-regulator of release probability. Nature 410, 41–49 (2001).

12. Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994).

13. Xu, J., Mashimo, T. & Südhof, T.C. Synaptotagmin-1, -2, and -9: Ca2+-sensors for fast release that specify distinct presynaptic properties in subsets of neurons. Neuron 54, 567–581 (2007).

14. Sun, J. et al. A dual Ca2+-sensor model for neuro-transmitter release in a central synapse. Nature 450, 676–682 (2007).

15. Cao, P., Maximov, A. & Südhof, T.C. Activity-dependent IGF-1 exocytosis is controlled by the Ca2+-sensor synaptotagmin-10. Cell 145, 300–311 (2011).

16. McMahon, H.T., Missler, M., Li, C. & Südhof, T.C. Complexins: cytosolic proteins that regulate SNAP-receptor function. Cell 83, 111–119 (1995).

17. Maximov, A., Tang, J., Yang, X., Pang, Z. & Südhof, T.C. Complexin controls the force transfer from SNARE complexes to membranes in fusion. Science 323, 516–521 (2009).

18. Tang, J. et al. Complexin/synaptotagmin-1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175–1187 (2006).

19. Wang, Y., Okamoto, M., Schmitz, F., Hofman, K. & Südhof, T.C. RIM: a putative Rab3-effector in regulating synaptic vesicle fusion. Nature 388, 593–598 (1997).

20. Kaeser, P.S. et al. RIM proteins tether Ca2+-channels to presynaptic active zones via a direct PDZ-domain interaction. Cell 144, 282–295 (2011).

21. Schoch, S. et al. RIM1a forms a protein scaffold for regulating neurotransmitter release at the active zone. Nature 415, 321–326 (2002).

22. Verhage, M. et al. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287, 864–869 (2000).

 

SOURCE

http://www.nature.com/focus/Lasker/2013/pdf/ES-Lasker13-Sudhof.pdf

NATURE MEDICINE | SPOONFUL OF MEDICINE

Lasker Awards go to rapid neurotransmitter release and modern cochlear implant

09 Sep 2013 | 13:38 EDT | Posted by Roxanne Khamsi | Category: Neuroscience/mental health, Odds and ends

Posted on behalf of Arielle Duhaime-RossA very brainy area of research has scooped up one of this year’s $250,000 Lasker prizes, announced today: The Albert Lasker Basic Medical Research Award has gone to two researchers who shed light on the molecular mechanisms behind the rapid release of neurotransmitters—findings that have implications for understanding the biology of mental illnesses such as schizophrenia, as well the cellular functions underlying learning and memory formation.By systematically analyzing proteins capable of quickly releasing chemicals in the brain, Genentech’s Richard Scheller and Stanford University’s Thomas Südhofadvanced our understanding of how calcium ions regulate the fusion of vesicles with cell membranes during neurotransmission. Among Scheller’s achievements is the identification of three proteins—SNAP-25, syntaxin and VAMP/synaptobrevin—that have a vital role in neurotransmission and molecular machinery recycling. Moreover, Südhof’s observations elucidated how a protein called synaptotagmin functions as a calcium sensor, allowing these ions to enter the cell. Thanks to these discoveries, scientists were later able to understand how abnormalities in the function of these proteins contribute to some of the world’s most destructive neurological illnesses. (For an essay by Südhof on synaptotagmin, click here.)The Lasker-DeBakey Clinical Medical Research Award went to three researchers whose work led to the development of the modern cochlear implant, which allows the profoundly deaf to perceive sound. During the 1960s and 1970s Greame Clark of the University of Melbourne and Ingeborg Hochmair, CEO of cochlear implant manufacturer MED-EL, independently designed implant components that, when combined, transformed acoustical information into electrical signals capable of exciting the auditory nerve. Duke University’s Blake Wilson later contributed his “continuous interleaved sampling” system, which gave the majority of cochlear implant wearers the ability to understand speech clearly without visual cues. (For a viewpoint by Graeme addressing the evolving science of cochlear implants, click here.)Bill and Melinda Gates were also honored this year with the Lasker-Bloomberg Public Service Award. Through their foundation, the couple has made large investments in helping people living in developing countries gain access to vaccines and drugs. The Seattle-based Bill & Melinda Gates Foundation also runs programs to educate women about proper nutrition for their families and themselves. The organization has a broad mandate in public health; one of its most well known projects is the development of a low-cost toilet that will have the ability to operate without water.The full collection of Lasker essays, as well as a Q&A between Lasker president Claire Pomeroy and the Gateses, can be found here.

 http://blogs.nature.com/spoonful/2013/09/lasker-awards-go-to-rapid-neurotransmitter-release-and-modern-cochlear-implant.html

Summary

Author: Larry H Bernstein, MD, FCAP

Chapter IX focused on VSM of the artery and related the action of calcium-channel blockers (CCMs) to the presynaptic interruption of synaptic-vesicle fusion necessary for CA+ release that leads to neurotransmitter secretion.  Under the circumstance neurotransmitter activation, the is VSM contraction (associated with tone).  The effect of CCB action on neurotransmitter action, there is a resultant vascular dilation facilitating flow.    In this section, we extend the mechanism to other smooth muscle related action in various organs.

[1] Neural activation of vas deferens smooth muscle cells

Noradrenaline (NA) acts by stimulating α1-adrenoreceptors to produce InsP3, which then releases Ca2+ that may induce an intracellular Ca2+ wave similar to that triggered by the ATP-dependent entry of external Ca2+. In addition, the α1-adrenoreceptors also activate the smooth muscle Rho/Rho kinase signaling pathway that serves to increase the Ca2+ sensitivity of the contractile machinery.

[2]  Urinary bladder and micturition

The bladder, which functions to store and expel urine, is surrounded by layers of detrusor SMCs. The latter have two operational modes: during bladder filling they remain relaxed but contract vigorously to expel urine during micturition. The switch from relaxation to contraction, which is triggered by neurotransmitters released from parasympathetic nerves, depends on the acceleration of an endogenous membrane oscillator that produces the repetitive trains of action potentials that drive contraction.

SMCs are activated by membrane depolarization (ΔV) that opens L-type voltage-operated channels

This mechanism of activation is also shared by [1], and uterine contraction. SMCs are activated by membrane depolarization (ΔV) that opens L-type voltage-operated channels (VOCs) allowing external Ca2+ to flood into the cell to trigger contraction. This depolarization is induced either by ionotropic receptors (vas deferens) or a membrane oscillator (bladder and uterus). The membrane oscillator, which resides in the plasma membrane,  generates the periodic pacemaker depolarizations responsible for the action potentials that drive contraction.

The main components of the membrane oscillator are the Ca2+ and K+ channels that sequentially depolarize and hyperpolarize the membrane, respectively. This oscillator generates the periodic pacemaker   depolarizations that trigger each action potential. The resulting Ca2+ signal lags behind the action potential because it spreads into the cell as a slower Ca2+ wave mediated by the type 2 RYRs.   Neurotransmitters such as ATP and acetylcholine (ACh), which are released from parasympathetic axonal varicosities that innervate the bladder, activate or accelerate the oscillator by inducing membrane depolarization (ΔV).

[3] The depolarizing signal that activates gastrointestinal, urethral and ureter SMCs is as follows:

A number of SMCs are activated by pacemaker cells such as the interstitial cells of Cajal (ICCs) (gastrointestinal and urethral SMCs) or atypical SMCs (ureter). These pacemaker cells have a cytosolic oscillator that generates the repetitive Ca2+ transients that activate inward currents that spread through the gap junctions to provide the depolarizing signal (ΔV) that triggers contraction. Our greatest interest has been in this mechanism. The rhythmical contractions of vascular, lymphatic, airway and corpus cavernosum SMCs depend on an endogenous pacemaker driven by a cytosolic Ca2+ oscillator that is responsible for the periodic release of Ca2+ from the endoplasmic reticulum. The periodic pulses of Ca2+ often cause membrane depolarization, but this is not part of the primary activation mechanism but has a secondary role to synchronize and amplify the oscillatory mechanism. Neurotransmitters and hormones act by modulating the frequency of the cytosolic oscillator.

Vascular or airway SMCs are driven by a cytosolic oscillator that generates a periodic release of Ca2+ from the endoplasmic reticulum that usually appears as a propagating Ca2+ wave.

The following points are repeated:

Step 1. The initiation and/or modulation of this oscillator depends upon the action of transmitters and hormones such as ACh, 5-HT, NA and endothelin-1 (ET-1) that increase the formation of InsP3 and diacylglycerol (DAG), both of which promote oscillatory activity.

Step 2. The oscillator is very dependent on Ca2+ entry to provide the Ca2+ necessary to charge up the stores for each oscillatory cycle. The nature of these entry mechanisms vary between cell types.

Step 3. The entry of external Ca2+ charges up the ER to sensitize the RYRs and InsP3 receptors prior to the next phase of release.

The proposed role of cyclic ADP-ribose (cADPR) in airway SMCs is consistent with this aspect of the model on the basis of its proposed action of stimulating the SERCA pump to enhance store loading and such a mechanism has been described in colonic SMCs.

Step 4. The mechanism responsible for initiating Ca2+ release may depend either on the RYRs or the InsP3 receptors (I). RYR channels are sensitive to store loading and the InsP3 receptors will be sensitized by the agonist-dependent formation of InsP3.

The global Ca2+ signal then activates contraction

Smooth muscle cell calcium activation mechanisms. Berridge MJ.
J Physiol. 2008; 586(Pt 21):5047-61. http://dx.doi.org/10.1113/jphysiol.2008.160440

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