The Voltage-Gated Calcium Channel Functions as the Molecular Switch of Synaptic Transmission

BI82CH01-Atlas ARI 9 January 2013 21:27

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The Voltage-Gated CalciumChannel Functions as theMolecular Switch of SynapticTransmissionDaphne AtlasDepartment of Biological Chemistry, Institute of Life Sciences, The Hebrew University ofJerusalem, 91904 Jerusalem, Israel; email: [email protected]

Annu. Rev. Biochem. 2013. 82:1.1–1.29

The Annual Review of Biochemistry is online atbiochem.annualreviews.org

This article’s doi:10.1146/annurev-biochem-080411-121438

Copyright c© 2013 by Annual Reviews.All rights reserved

Keywords

amperometry, intramembrane signaling, chromaffin cells, evokedsecretion, excitosome, lanthanides

Abstract

Transmitter release is a fast Ca2+-dependent process triggered in re-sponse to membrane depolarization. It involves two major calcium-binding proteins, the voltage-gated calcium channel (VGCC) and thevesicular protein synaptotagmin (syt1). Ca2+ binding triggers trans-mitter release with a time response of conformational changes thatare too fast to be accounted for by Ca2+ binding to syt1. In con-trast, conformation-triggered release, which engages Ca2+ binding toVGCC, better accounts for the fast rate of the release process. Here,we summarize findings obtained from heterologous expression sys-tems, neuroendocrine cells, and reconstituted systems, which revealthe molecular mechanism by which Ca2+ binding to VGCC triggersexocytosis prior to Ca2+ entry into the cell. This review highlights themolecular aspects of an intramembrane signaling mechanism in whicha signal is propagated from the channel transmembrane (TM) domainto the TM domain of syntaxin 1A to trigger transmitter release. It dis-cusses fundamental problems of triggering transmitter release by syt1and suggests a classification of docked vesicles that might explain syn-chronous transmitter release, spontaneous release, and facilitation oftransmitter release.

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Review in Advance first posted online on January 17, 2013. (Changes may still occur before final publication online and in print.)

Changes may still occur before final publication online and in print

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Contents

OVERVIEW . . . . . . . . . . . . . . . . . . . . . . . 1.2INTRODUCTION. . . . . . . . . . . . . . . . . 1.3

The Kinetics of Exocytosis . . . . . . . . 1.3The Current Model . . . . . . . . . . . . . . 1.3Synaptotagmin is the Calcium

Sensor of Secretion: TheCurrent Model . . . . . . . . . . . . . . . . 1.4

Could Synaptotagmin 1 Accountfor the Speed of the ReleaseProcess? . . . . . . . . . . . . . . . . . . . . . . 1.5

PROPOSED MODEL OFEVOKED TRANSMITTERRELEASE . . . . . . . . . . . . . . . . . . . . . . . 1.6The VGCC is the Calcium Sensor

of Secretion: Rationale. . . . . . . . . 1.6Physical and Functional

Interactions of VGCC with thet-SNAREs Syntaxin 1A andSNAP-25 and the v-SNARESynaptobrevin . . . . . . . . . . . . . . . . . 1.6

The Excitosome, a HeteroproteinComplex Serving asan Exocytosis Unit . . . . . . . . . . . . 1.7

Classification of Docked Vesiclesas Releasable and NonreleasableVesicles . . . . . . . . . . . . . . . . . . . . . . . 1.8

THE VGCC IS AVOLTAGE-SENSITIVECALCIUM-BINDINGPROTEIN. . . . . . . . . . . . . . . . . . . . . . . 1.9

THE VGCC IS THE CALCIUMSENSOR OF SECRETION:TESTING THE PROPOSEDMODEL. . . . . . . . . . . . . . . . . . . . . . . . . 1.10

Impermeable Cations Bound at theChannel Selectivity FilterSupport Evoked Secretion . . . . . 1.10

Impermeable VGCC SupportsEvoked Secretion. . . . . . . . . . . . . . 1.12

MOLECULAR MECHANISM OFVGCC: RECONSTITUTIONOF DEPOLARIZATION-EVOKED TRANSMITTERRELEASE . . . . . . . . . . . . . . . . . . . . . . . 1.13

HOW DOES THE VGCCTRIGGERNEUROTRANSMITTERRELEASE? . . . . . . . . . . . . . . . . . . . . . . 1.15The Channel Pore Triggers

Evoked Secretion by Switchingfrom the Nonconductive to theConductive Mode . . . . . . . . . . . . . 1.15

Transmission of the Signal fromthe Channel to the ExocytoticMachinery via Syntaxin 1A . . . . . 1.16

THE CURRENT MODELVERSUS THE EXCITOSOMEMODEL. . . . . . . . . . . . . . . . . . . . . . . . . 1.16

SPONTANEOUS RELEASE ASVIEWED BY THE CURRENTMODEL AND THEEXCITOSOME MODEL. . . . . . . . 1.18Spontaneous Release According to

the Current Model . . . . . . . . . . . . 1.18Spontaneous Release According to

the Excitosome Model . . . . . . . . . 1.18WHAT MAKES THE VGCC

AN ATTRACTIVE CALCIUMSENSOR OF SECRETION? . . . . 1.19

CONCLUDING REMARKS . . . . . . . 1.20

OVERVIEW

Numerous studies have described multiplemechanisms of transmitter release driven by avariety of signals (e.g., membrane depolariza-tion, caged calcium, ionomycin, the Gq-PLC-IP3 pathway, caffeine) (1–4). However, manyfundamental issues are unresolved, particularly

with regard to the primary event that underliesaction potential–driven synaptic transmission.The overall goal of this review is to highlightthe role of Ca2+ binding at the voltage-gatedcalcium channel (VGCC) in signaling exocyto-sis, focusing mainly on the mechanism of secre-tion induced by membrane depolarization. The

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VGCCs:voltage-gated calciumchannels

Excitosome complex:a heteroprotein

complex consisting ofthe channel, Sx1A,SNAP-25, and syt1

SNARE: solubleN-ethylmaleimide-sensitive factorattachment proteinreceptors

review discusses the excitosome model, inwhich the channel is assembled into the het-eroprotein excitosome complex by syntaxin 1A(Sx1A), synaptosome-associated protein 25 kDa(SNAP-25), and synaptotagmin (syt1), and thevesicles are docked to the plasma membranevia unprimed (Ca2+-unbound syt1) or primed(Ca2+-bound syt1) excitosome complexes.

In this model, the channel operates as a Ca2+

sensor that triggers secretion while syt1 func-tions as a vesicle-priming protein. The currentmodel suggests that syt1 is the Ca2+ sensor ofsecretion and the channel is the vehicle that in-troduces Ca2+ into the cell. We examine themolecular events underlying the coordinated,regulated process that resolve the Ca2+ bind-ing site of the VGCC as the initial trigger ofsecretion. The proposed model identifies syt1as a central player in vesicle priming and pro-motes the VGCC as the putative Ca2+ sensorof secretion.

INTRODUCTION

The Kinetics of Exocytosis

Synaptic transmission is a highly regulatedprocess that closely links depolarization-evoked Ca2+ channel opening to vesicle fusionon a submillisecond timescale. Similar to theexcitation-contraction coupling in muscle cells,exocytosis, or excitation-secretion coupling, isunder tight spatial and temporal control. TheCa2+-dependent event called exocytosis is oneof the most rapid biological processes. It istriggered in ∼100 μs in the central synapse ofneuronal cells and in ∼1 ms in medullary chro-maffin or pancreatic β cells (2, 3, 5–10). Katz& Miledi’s (11) initial studies of exocytosis in-dicated the major role of Ca2+ in the initiationof the release process. The authors describedthe common presynaptic mechanism thus:“The utilization of external calcium ions at theneuromuscular junction is restricted to a briefperiod which barely outlasts the depolarizationof the nerve ending, and which precedes thetransmitter release itself” (11, p. 535).

A subsequent interpretation of the calciumhypothesis of Katz & Miledi was that depolar-

ization increases the calcium conductance ofthe nerve terminal membranes, and the Ca2+

coming through the open calcium channel bindto a presynaptic protein that transiently in-creases the probability of quantal release. Ac-cordingly, the prevailing dogma promotes theview that neurotransmitter release is triggeredsubsequent to Ca2+ inflow by Ca2+ binding toan intracellular/vesicular protein(s). The highspeed of synaptic transmission requires a closeassociation of the participating proteins. Anelectron microscope tomography study esti-mated ∼20 nm for the distance between presy-naptic Ca2+ channels and synaptic vesicles at theactive zone of the frog neuromuscular junction(12). Evidence for the existence of nanodomaincoupling, in which the VGCC and the synap-tic proteins are in close association, suggest onthe one hand a means of facilitating Ca2+ dif-fusion and on the other a close proximity andfunctional interaction of the channel with theexocytotic machinery. Both could explain thefast rate and tight regulation of exocytosis (1–4,13–19).

The Current Model

According to the current model, synaptic vesi-cle fusion involves a family of proteins termedSNAREs (soluble N-ethylmaleimide-sensitivefactor attachment protein receptors) (20, 21).They form the central part of the complexsystem of synaptic neurotransmission, whichtogether with syt1 triggers secretion (2, 22).These highly conserved proteins also partic-ipate in the mechanisms of intracellular pro-tein transport (20, 23, 24) and are consideredSNARE engines for membrane fusion (25). Thepresynaptic plasma membrane SNARE pro-teins (t-SNAREs or Q-SNAREs) are Sx1A,SNAP-25, and VAMP2 (vesicle-associatedv-SNARE or R-SNARE, also called synapto-brevin). These proteins are selective substratesof Clostridium botulinum toxins botulinum C1(BotC1; cleaves Sx1A), botulinum A and bo-tulinum E (BotA and BotE, respectively; bothcleave SNAP-25), and tetanus toxin (TetX;cleaves VAMP2). These neurotoxins function

www.annualreviews.org • The VGCC is the Ca2+ Sensor of Secretion 1.3


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as zinc proteases that abolish synaptic trans-mission through selective proteolysis of theSNARE proteins (26–29).

According to the SNARE model, throughforming a tight coiled-coil complex, theSNAREs assemble in trans between the vesi-cle and plasma membranes, overcoming themembrane-energy barrier and triggering vesi-cle fusion. Both the v-SNARE and t-SNAREsmediate the fusion itself according to this model(but see References 30 and 125). Upon arrivalof an action potential, the VGCC opens andthere is an increase in local cytosolic Ca2+ con-centrations. Ca2+ binds to the vesicle-anchoredsyt1 and triggers release by inducing a fast si-multaneous interaction of syt1 with the SNAREcomplexes and phospholipids (31).

Additional proteins involved in the fusionevent are Munc18-1, Munc13-1, and com-plexin. Initially, Munc18-1 is bound to theclosed conformation of Sx1A. Docking of asynaptic vesicle results in a tripartite tether-ing complex consisting of Rab3, RIM1 (Rab3-interacting molecule 1), and Munc13-1, whichis supposed to catalyze the conversion ofSx1A into an open conformation. Then, theSx1A/SNAP-25 binary t-SNARE complex as-sembles with VAMP2 to form the SNAREpin(3). The SNAREpins are complexes ofSNAREs that spontaneously assemble into astable (32) four-helix bundle (33). This com-plex, which is formed between membranes asa trans-SNARE complex, catalyzes fusion byforcing membranes closely together as it zip-pers up, exerting force against the separationof its helices from each other. The SNARE-pin binds complexin, which acts like a fusionclamp by preventing the completion of SNAREcomplex zippering. Complexin, a Ca2+-bindingprotein, operates in conjunction with syt1by controlling trans-SNARE-complex assem-bly (34), and upon sensing an increase in thelocal Ca2+ concentration, triggers fusion-poreopening (35). More recent reports showed thatcomplexin’s main activity is in priming andclamping synaptic exocytosis (36). Although theSNAREs and their assembly into the SNARE-pin complex are crucial for vesicle fusion, the

mechanism by which syt1 regulates SNARE-catalyzed fusion remains poorly understood.

Synaptotagmin is the Calcium Sensorof Secretion: The Current Model

According to the current model, the vesicularprotein syt1 acts as a major Ca2+ sensor forneuronal exocytosis (2, 22, 25, 31, 37, 38). syt1contains a single transmembrane (TM) domainfollowed by a cytoplasmic domain consistingof two vicinal C2 domains, C2A and C2B (37),similar to those observed in protein kinase C(PKC) (39). The cytoplasmic domain of syt1binds acidic phospholipids, indicating an inter-action with the hydrophobic core and the headgroups of membrane phospholipids.

Ca2+ binds to the top loops of the C2 do-mains, which form specific sites for two to threeCa2+ ions in close vicinity (40). Binding of Ca2+

to syt1 C2A and C2B domains does not inducelarge conformational changes in the protein.This unique property led researchers to sug-gest that syt1 acts like an electrostatic switch,whereby changes in electrostatic potential trig-ger interactions with target molecules (41, 42).

Both C2 domains bind to the SNARE pro-teins (15, 43, 44), to phospholipids in a Ca2+-dependent manner (45), and to VGCC (14–17,46). The Ca2+-dependent phospholipid bind-ing to the C2 domains is similar to that of pro-teins such as rabphilin, PKC, and phospholipaseC (47–50). The binding of the C2 domains tophospholipid membranes seems integral to syt1function. syt1 binds to anionic lipids both inthe absence of Ca2+, through a polybasic patchconsisting of four lysine residues on the C2Bdomain, and through its Ca2+ binding sites (25,51–53).

syt1 also binds to VGCC through thepolylysine motif at the C2A domain (46). Thispolybasic motif interacts with a cytosolic do-main that links segments II and III of Cav1.2,Cav2.1, Cav2.2, or Cav2.3, and is critical forCa2+-independent interactions of VGCC withsyt1 (14–16, 54, 55). A mutated polylysine mo-tif abolishes C2A binding to Cav1.2 (46), de-creases evoked secretion in PC12 cells (56), and

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Spontaneous release:release of

neurotransmitterquanta at a lowfrequency triggered bystochastic events

abolishes depolarization-evoked capacitancetransients in reconstituted secretion (129). Thepolylysine motif of C2B is required for efficientCa2+-independent docking and/or priming ofsynaptic vesicles in vivo (57).

The basic residues in C2B domains, posi-tioned away from the Ca2+ binding site, bindphosphatidylinositol 4,5-bisphosphate (51).These basic residues contribute to membraneaggregation in vitro and could exert pullingforces between apposing membranes, facilitat-ing vesicle fusion. The current model of exo-cytosis also suggests a specific role for the hy-drophobic residues at the tip of the syt1 Ca2+

binding site. Accordingly, these residues pen-etrate the anionic membranes (58) and pro-mote fusion through induction of membranecurvature (59–61; see Reference 62 for a re-view). Hence, Ca2+-bound syt1 acts by increas-ing the membrane tension, locally pulling thetwo membranes to a close distance where the in-crease in membrane curvature facilitates vesiclefusion (60, 61).

Fernandez-Chacon et al. (63) provided themajor proof that established syt1 as the Ca2+

sensor of neurotransmission at the synapse (seeThe Current Model Versus the ExcitosomeModel, below). In their study, point mutationin syt1 basic residue R233Q, close to the Ca2+

binding domain of C2A, caused a twofolddecrease in overall Ca2+ affinity. When in-troduced into the endogenous synaptotagmin Igene in mice, this point mutation reducedthe probability of synaptic release, decreasingthe Ca2+ sensitivity of synchronous neuro-transmitter release twofold without affectingspontaneous release or the size of the readilyreleasable pool of synaptic vesicles. In laterstudies, the R233Q mutation equally decreasedspontaneous release (22, 31, 64).

Although these studies considered syt1 theCa2+ sensor of secretion, the multiple stepssubsequent to membrane depolarization—including Ca2+-channel gating, intracellularCa2+ diffusion, Ca2+ binding to vesicularSNARE/assembled proteins, membraneinsertion of syt1 C2 domains, and vesiclefusion—could not account for the 60–100 μs

onset of depolarization-evoked transmitterrelease. In this model, the VGCC servesexclusively as a vehicle to introduce Ca2+ intothe cell, and the potential role of Ca2+ bindingto VGCC in triggering synaptic transmissionis completely ignored.

Could Synaptotagmin 1 Account forthe Speed of the Release Process?

Ca2+-unbound syt1 is repelled from the mem-brane by the C2A-C2B negative charges, whichhold the vesicle at some distance away fromthe membrane (58, 65). Ca2+ neutralization ofthe negative charge of the C2AB pockets is es-sential for activating the electrostatic switch(66). Only the change in the net electrostaticcharge provides for the interactions of the pos-itively charged residues at the tips of the C2ABpockets with the negatively charged presynapticmembrane, allowing syt1 insertion in the mem-brane (25, 41, 58, 65, 67). This change, which isalso required for promoting new interactions ofsyt1 with the negatively charged regions of theSNAREs and other presynaptic proteins withinthe fusion machinery prior to vesicle fusion,emerges as an intermediary step in the fusionevent. Thus, the preassembly of the vesicle ina ready-to-go high-energy state (2, 45, 68, 69)is achieved by diminishing the electrostatic gapwith the membrane phospholipids and bring-ing the vesicle to a close apposition with themembrane (70).

In vivo studies in Drosophila show that Ca2+

binding at the syt1 C2 domains is essential formembrane insertion (58, 65). The decrease inCa2+ affinity of sytB−RQ, a mutation at the syt1C2B domain that impairs syt1 insertion in themembrane, decreases Ca2+-evoked transmitterrelease (65). These in vivo studies reveal thatin the absence of Ca2+, syt1 holds the vesi-cle away from the membrane, and only theCa2+-bound C2AB domain can dock the vesi-cles close enough to allow SNARE complex for-mation and membrane fusion (25). Similarly, invitro studies show that in the absence of Ca2+,the polybasic patch on the C2B domain tethersthe liposomes, but electrostatic repulsion keeps



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Conformationalchange: a change inthe shape of amolecule that couldresult from a change intemperature, pH,voltage, ionconcentration,phosphorylation, orligand binding

them too far apart for SNARE complexes toform (25).

It appears that subsequent to Ca2+ bindingto syt1 and membrane insertion, a multitudeof preparatory reactions is required prior tofacilitating vesicle fusion. The time frame fromthe initial Ca2+ binding to syt1, mainly throughC2 domain insertion at the membrane, throughthe initiation of interactions with the SNAREs,binding to phospholipids, and subsequentlowering of the activation energy for fusion,exceeds the submilliseconds range of evokedrelease. Membrane insertion of the cytosolic C2

domain of PKCγ-GFP lasts many seconds (71,72). The rather slow process of C2 insertionin the membrane implies that Ca2+ binding tosyt1 is an intermediate step in the fusion event,designating syt1 as a vesicle-priming protein(1, 76). The above considerations challengesyt1 as the Ca2+ sensor of secretion and furtherimply the involvement of a submillisecondfusion-triggering Ca2+ sensor protein.

PROPOSED MODEL OF EVOKEDTRANSMITTER RELEASE

The VGCC is the Calcium Sensor ofSecretion: Rationale

The timescale of Ca2+-dependent secretoryevents varies from synaptic transmission to neu-roendocrine transmission, ranging from sub-milliseconds to a few milliseconds. Bruns &Jahn (73) showed that release from eithersmall clear (synaptic) or large dense core vesi-cles (chromaffin granules) initiated rapidly witha rise time of less than 60 μs, and con-tent discharge occurred with time constants of260 μs (synaptic vesicles) and 1.3 ms (chro-maffin granules). These rates typical of con-formational changes suggest that exocytosis isa conformation-triggered event. Accordingly,a Ca2+-dependent conformational change trig-gers the fusion of a high-energy-state vesicle ona submillisecond timescale.

The excitosome model proposes that:

1. he VGCC functions as a Ca2+ sensor pro-tein of secretion.

2. Secretion is activated by a conformationalchange triggered during Ca2+ binding atthe open channel pore, prior to Ca2+

entry.3. The conformational change coupled to

cation binding at the pore propagatesdirectly to the assembled ready-to-goprimed vesicle at the nerve terminal.

4. Termination of secretion occurs whenthe channel returns to the closed-adaptedstate.

5. The submillisecond opening of the chan-nel and the ensuing conformationalchanges are transmitted via intramem-brane signaling through the TM domainsto the exocytotic machinery prior to Ca2+

entry, accounting for the rapid time frameof evoked release (1, 74–77).

Physical and Functional Interactionsof VGCC with the t-SNAREs Syntaxin1A and SNAP-25 and the v-SNARESynaptobrevin

Exocytosis triggered by a conformationalchange requires the close association of thechannel with the exocytotic proteins. Earlycoimmunoprecipitation studies showed thatmonoclonal antibodies for Sx1A (HPC-1)and anti-syt1 antibodies recognize the ω-conotoxin-sensitive N-type calcium channelsolubilized from rat brain (43, 78). Consistentwith these data, colocalization of calcium en-try sites and exocytotic release sites was shownin adrenal chromaffin cells (79) and in mousepancreatic β cells (80).

A physical association of the N-type VGCC(Cav2.2) with Sx1A and syt1 was furtherconfirmed by identifying the intracellular looplinking segments II and III of Cav2.2 as thesite of interaction with Sx1A (15, 16, 18, 77,81–86). These direct binding assays utilizethe recombinant intracellular II-III loop ofCav2.2 and the cytosolic SNAP-25, syt1 C2Aand syt1 C2B, or the N terminus of Sx1A(87). In parallel, research demonstrated in theheterologous Xenopus oocyte expression systemfunctional modulation of the kinetic properties

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Nonreleasablevesicle: a dockedvesicle tethered to themembrane via anonprimedexcitosome, in whichsyt1 is not bound toCa2+ and not insertedin the presynapticmembrane

Releasable vesicle: adocked vesicletethered to themembrane via aprimed excitosome, inwhich syt1 is Ca2+bound and fullyinserted at thepresynaptic membrane

of Cav1.2 (L-type), Cav2.2, Cav2.3 (R-type),and Cav2.1 (P/Q-type) channels by the synapticproteins (14, 15, 88; for reviews, see 1, 18).Full-length VGCC, Sx1A, SNAP-25, andsyt1 are readily expressed in the oocytes andtargeted to their cellular site while maintain-ing their native properties. Protein-proteininteractions revealed in oocytes are reliablyquantified using the standard two-electrodevoltage-clamped method (88). Interactions arescreened by the kinetic parameters of channelactivation, voltage-dependent inactivation,voltage-dependent steady-state inactivation, orcurrent amplitude (77, 89).

The oocyte expression system allows a sys-tematic study of cardiac (Cav1.2) or neuronalCav2.2, Cav2.3, and Cav2.1 channels’ interac-tions with synaptic proteins (13, 81, 83, 88,90–93). It also enables recording of VGCC ki-netics, monitoring interaction with Sx1A mu-tants (74) and syt1 mutants (46, 75) as well asexploring BotC1, BotE, BotA, and TetX ef-fects on synaptic proteins/channel interactions(54, 74, 92). Furthermore, studies in BAPTA-injected oocytes monitor in great detail theCa2+-independent functional interactions be-tween the channel and the synaptic proteins (seebelow). These studies have provided evidencefor tight bidirectional interactions of the chan-nel with the exocytotic proteins. Use of interac-tions of full-length proteins examined at theirnative location in the cell has a major advantageover the use of truncated recombinant proteinsin various in vitro systems.

The Excitosome, a HeteroproteinComplex Serving as an Exocytosis Unit

Initial immunoprecipitation experiments sug-gested that a population of the N-type chan-nel (Cav2.2) associates with Sx1A and syt1 toform a ternary complex (78). A distinct voltage-dependent steady-state inactivation profilerecorded in voltage-clamped oocytes revealeda functional putative complex, assembled byCav2.2 or Cav1.2 with Sx1A, SNAP-25, andsyt1, and named an excitosome (14). Subse-quent studies confirmed that VGCCs (e.g.,

Cav2.2, Cav1.2, Cav2.3, or Cav2.1) associatewith Sx1A, SNAP-25, and syt1, forming func-tional and kinetically distinct heteroproteincomplexes (Figure 1) (14, 54, 55).

The deceleration of Cav1.2 activation andthe decrease in current amplitude inducedby the binary Sx1A/SNAP-25 complex coex-pressed in oocytes are restored with syt1, dis-closing a tripartite interaction of Sx1A/SNAP-25/syt1 and a reciprocity with the channel (14,15). These channel/synaptic protein interac-tions monitored in BAPTA-injected oocytesimply that the formation of the excitosomecomplex is Ca2+ independent, and syt1 C2ABare Ca2+ free. The assembly of the unprimedexcitosome by essential prefusion steps enablesvesicle tethering to the membrane but does notallow syt1 insertion into the membrane (seesidebar, Docked Vesicles According to the Ex-citosome Model) (46). Ca2+-independent stoi-chiometric interactions of syt1 C2 domains andthe t-SNARE heterodimer were also shown us-ing purified proteins from brain membranes(94).

Ca2+ binding to syt1 within the excitosomecomplex may serve to promote new interac-tions with the synaptic proteins and/or thechannel. In doing so, proteins rotating rela-tive to one another may move closer or fartherapart from one another. Alternatively, Ca2+-bound syt1 may act by simply disrupting exist-ing Ca2+-independent intermolecular protein-protein interactions with other members of thecomplex. Thus, the significant changes at syt1during Ca2+ binding at the C2 domains (2, 22,95, 96), in addition to insertion into the mem-brane, transform a nonreleasable vesicle teth-ered to the membrane with Ca2+-unbound syt1to a releasable vesicle tethered to the mem-brane via Ca2+-bound syt1 (primed excitosome)(see sidebar, Docked Vesicles According to theExcitosome Model) (1, 54). The term primingdescribes the steps necessary for a VGCC/t-SNAREs/syt1-tethered vesicle docked via anunprimed excitosome to become ready for fu-sion through Ca2+ binding to syt1 C2AB sites,i.e., generating a primed excitosome. The exci-tosome complex as an exocytotic unit is tailored



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Nonprimed excitosomesyt1(–Ca), SNAP-25,

syntaxin 1A, Ca2+ channel

Nonreleasable vesiclesReleasable vesicles

At any given time Ca2+-free or Ca2+-bound syt1vesicles are tethered to the channel

via the excitosome complex

Physiological stimulation

Active zone

Action potential

Primed excitosomesyt1(+Ca), SNAP-25,

syntaxin 1A, Ca2+ channel

Figure 1Docked vesicles, classified as nonreleasable and releasable vesicles, are tethered to the membrane via the excitosome complex. At anygiven time, some of the vesicles present at the terminal have already encountered Ca2+ and therefore are tethered to the membrane viathe primed excitosome complex composed of Ca2+-bound syt1 (synaptotagmin). These are called the releasable vesicles. Nonreleasablevesicles are tethered to the membrane via a nonprimed excitosome, composed of Ca2+-free syt1. They are positioned at some distanceaway from the membrane owing to the electrostatic repulsion between Ca2+-unbound syt1 and the membrane phospholipids. Uponarrival of an action potential, the releasable vesicles release their content in a submillisecond time frame. At the same time, through[Ca2+]i rise and binding to syt1, the nonreleasable vesicles are primed and join the releasable pool. Hence, the action potential, on onehand, releases the releasable, and on the other, primes the nonreleasable. The ratio between the releasable and nonreleasable poolsdetermines the robustness of evoked secretion. This ratio varies among terminals of various neuronal and neuroendocrine cells.

to provide spatial and temporal control of therelease process, securing a close functional as-sociation of the vesicle, VGCC, and synapticproteins at the nerve terminal.

This classification of the docked vesiclesuggests that the primed vesicles, tetheredby the primed excitosome, fuse with themembrane in response to a conformationalsignal transmitted from the channel poreprior to Ca2+ entry. Conversely, vesicles thatare tethered at some distance away from themembrane via the unprimed excitosome needto be primed first through Ca2+ binding to the

syt1 C2AB pockets during [Ca2+]i rise. Hence,syt1 is a priming protein that controls the ratioof releasable/nonreleasable pools of vesicleswithin the excitable cell (see sidebar, DockedVesicles According to the Excitosome Model)(Figure 1).

Classification of Docked Vesicles asReleasable and Nonreleasable Vesicles

The cell displays a distinct fraction of its re-leasable and nonreleasable vesicles that main-tain synaptic communication during ongoing

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DOCKED VESICLES ACCORDING TO THE EXCITOSOME MODEL

Although synaptic vesicles are identical both biochemically and under the electron microscope, different types ofvesicles have been described as readily releasable, reserve, exo-endo recycling, immediately releasable, reluctant,resting, etc. More recently, research demonstrated that vesicles at the resting terminal could be classified intotwo major pools. The reserve pool, representing ∼90% of all vesicles, consists of vesicles somewhat removedfrom immediate secretion events. The remaining 10% consist of docked vesicles, which are actively involved inneurotransmitter release (97, 98).

The excitosome model suggests that docked vesicles are tethered to the membrane through a nonprimed orprimed excitosome complex (14, 16). syt1 is a member of the complex that can be in either a Ca2+-bound or aCa2+-unbound form, differentiating a releasable pool with syt1 (+Ca) from a nonreleasable pool with syt1 (–Ca)(Figure 1; see below). The nerve terminals normally display a distinct and typical ratio of releasable to nonreleasablepools of docked vesicles.

Nonreleasable Docked Vesicles

Nonreleasable vesicles are docked vesicles tethered to the membrane through protein-protein interactions withCa2+-free syt1, Sx1A, SNAP-25, and VGCC. The negative charge of the C2 syt1 Ca2+-binding pockets preventsinteractions of the positively charged amino acids at the tips of the C2 domains with phospholipids of the presynapticmembrane by electrostatic repulsion. Held at a physical distance from the anionic membrane, these docked vesicleswill not fuse during an action potential. The nonreleasable vesicles first need to be primed subsequent to Ca2+

binding to syt1, and syt1 has to insert at the bilayer (58, 65; see also Reference 25). These vesicles will not fuseduring an action potential (Figure 1).

Releasable Docked Vesicles

Releasable docked vesicles are tethered to the membrane via a primed excitosome, in which syt1 is Ca2+ boundand fully inserted at the presynaptic membrane. In this state, the preparatory interactions within the excitosomeare complete, and the vesicle is ready to fuse with the membrane. A conformational change propagated from theCa2+-occupied channel pore during an incoming action potential will trigger secretion from the releasable pool.

The Ca2+-bound state of syt1 C2 sites controls the proportion of releasable/nonreleasable vesicles that character-izes nerve terminals. The proportion of releasable/nonreleasable vesicles is unique and dictates the propensity of theterminal to undergo exocytosis and determines the robustness of secretion. For example, enlarging the Ca2+-boundsyt1 releasable pool by [Ca2+]i rise during repetitive stimulation facilitates vesicle fusion.

activity. Docked vesicles are tethered to the cellmembrane via the excitosome complex, whichconsists of VGCC, Sx1A, SNAP-25, and syt1.Resolved by the Ca2+ occupancy of syt1 C2ABsites, vesicles tethered to the membrane withCa2+-empty syt1 C2AB sites are nonreleasablevesicles, and those tethered with Ca2+-boundsyt1 are releasable vesicles (see sidebar, DockedVesicles According to the Excitosome Model).Excitosome complexes generated by differ-ent channels display different release kinetics,

manifested for example by catecholamine re-lease mediated by Cav1.2 in neuroendocrinecells, compared with synaptic transmission me-diated by Cav2.1, Cav2.2, or Cav2.3 in neuronalcells (46, 99).

THE VGCC IS AVOLTAGE-SENSITIVECALCIUM-BINDING PROTEIN

The Ca2+ binding site of VGCC is composedof four glutamate residues in the same relative



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Lanthanides: rareearth elements thatform trivalent cations,Ln3+, some of whichmimic Ca2+ activity

Ionic radius: thedistance between thecenter of the nucleusand the outer edge ofthe ion

Amperometry: atechnique that uses acarbon-fiber electrodebrought into closeproximity of the cellthat recordsamperometric spikesoriginating from theoxidation ofmonoamine moleculesreleased during thefusion of individualsecretory vesicles

position in the P loops of TM domains I to IVof the Ca2+ channel (Figure 2a,b) (100). Thiswell-defined EEEE Ca2+ binding site at thepore participates in the formation of the chan-nel selectivity filter (101) and can bind a singleCa2+ ion with high affinity (Kd 1 μM) and multi-ple ions with low affinity (Kd = 13.4 mM) (101–104). Kuo & Hess (103) proposed that a sin-gle occupancy of the high-affinity site (<1 μMCa2+) of the selectivity filter is responsible forblocking the channel, preventing Na+ flow.

Global conformational changes during theactivation of the voltage sensor of the chan-nel open the channel pore and expose its low-affinity Ca2+ binding site. It is commonly ac-cepted that the interaction of cations binding tothe negative glutamates of the pore determinesboth the conductivity and selectivity of the cal-cium channel. Membrane depolarization thatopens the channel pore provides initial confor-mational changes that allow binding of multi-ple cations. These changes at the pore combinewith changes induced by cations seeking out anoptimal interaction within the closely packedcarboxylate groups of the glutamate residues.In fact, this combined action represents a spe-cific example for the coupling of voltage sensormovement to the pore gate domain. The oc-cupancy of the selectivity filter by at least twocations allows rapid Ca2+ inflow, converting thenonconductive channel pore into a conductiveone (Figure 2a,b) (101).

THE VGCC IS THE CALCIUMSENSOR OF SECRETION:TESTING THE PROPOSEDMODEL

Two independent studies capture the effects onsecretion of cation binding at the channel poreprior to successive cation inflow. The first ex-amined cations that bind at the channel pore butare excluded from entry into the cell (105–108).The second utilized a Ca2+-impermeable chan-nel mutant that retained both its voltage sen-sitivity and Ca2+-binding capacity at the pore(Figure 2) (76, 109).

Impermeable Cations Bound at theChannel Selectivity Filter SupportEvoked Secretion

Lanthanides are rare earth metal ions that sharemany common Ca2+ binding sites on vari-ous enzymes, channels, and receptors. La3+, amember of the lanthanide family with a sim-ilar ionic radius (1.06 A) to Ca2+ (0.99 A),binds to the EEEE locus, the Ca2+ binding siteat the channel pore (101). Other members ofthe trivalent lanthanide family, cerium (Ce3+),presodymium (Pr3+), neodymium (Nd3+), eu-ropium (Eu3+), and gadolinium (Gd3+), alsocompete for binding to the channel pore. Theyare excluded from permeation through thechannel pore and provide a suitable means fortesting the ability of cation binding at the chan-nel to support evoked secretion, independent ofbinding to intracellular proteins.

Membrane depolarization in bovine chro-maffin cells triggered catecholamine secretionwhen La3+ was substituted for Ca2+ as thecharge carrier. Secretion was monitored byamperometry in conjunction with fura-2 flu-orescence imaging (105). La3+ also supporteddepolarization and high glucose–triggeredinsulin secretion in insulinoma cells (INS-1E)and pancreatic islets (106). Inhibition of La3+-mediated secretion by blocking the L-typechannel with nifedipine indicates that La3+

mediates secretion via binding to the L-typechannel (Figure 2c) (110). Lanthanides ofionic radius slightly smaller than La3+, suchas Ce3+ (1.034 A) and Pr3+ (1.013 A), supportdepolarization-evoked [3H]-serotonin releasefrom PC12 cells. However, the smaller ionicradii cations Eu3+ (0.95 A) and Gd3+ (0.938 A),which are known to bind at the pore, are un-able to substitute for the physiological activityof Ca2+ (105). The distinct size-restrictedsecretion process indicates involvement of awell-defined calcium binding site at the chan-nel pore and suggests an interaction of boundcation with atoms lining the channel pore.

Triggering of secretion by La3+ entryinto the cell is unlikely because La3+ currentsare not recorded in either patch-clamped

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a b

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e Ca2+ channel

Syntaxin 1A Syntaxin 1A CC/VV

Figure 2The calcium binding site of the voltage-gated calcium channel (VGCC) called the EEEE locus propagates asignal to syntaxin 1A (Sx1A). (a) Conversion of the selectivity filter of the VGCC from the nonconductingsingle-ion pore conformation to (b) a conducting multi-ion pore conformation involves a change in poreconformation. The model predicts that the conformational change that transforms the nonconducting poreto a conducting one triggers transmitter release when propagated to synaptic proteins associated with thechannel. (c) La3+ binding at the pore of the channel is excluded from cell entry but binds at the channel pore,substituting for Ca2+ as a charge carrier. La3+ bound at the channel pore appears to enable a conformationalchange at the channel pore that when propagated to the exocytotic machinery triggers transmitter release(d ) A single-point mutation (L775P) at the α11.2 pore-forming subunit of the L-type channel (Cav1.2)blocks Ca2+ permeation without affecting voltage sensitivity or Ca2+ binding at the channel pore. Theα11.2/L775P mutant supports transmitter release independently of Ca2+ entry. (e) Intramembrane signalingthrough the transmembrane (TM) domains of the channel pore and Sx1A was revealed by mutating twohighly conserved vicinal cysteine residues, C271V and C272V, at the Sx1A TM domain. The Sx1A mutantloses its negative modulation of current amplitude (74, 128) and abolishes evoked secretion in a reconstitutedsystem (75, 99).



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chromaffin cells or in oocytes expressing highlevels of recombinant Cav1.2 (105). Also,fura-2 fluorescence, which easily detects 1–5pM La3+ concentrations (111), shows nochange in intracellular La3+. These results areconsistent with the view that La3+ support ofevoked release is mediated though binding atthe channel pore and not though [La3+]i rise,which in permeabilized cells requires >1 μMLa3+ to trigger release (112).

The selective Cav1.2 channel agonists (e.g.,BayK-8644, FPL-64176) accelerate the initialand the sustained rates of catecholamine secre-tion in bovine chromaffin cells using Ca2+ orLa3+ as the charge carrier (108). The similarincrease in the extent of agonist-induced cate-cholamine secretion further supports the viewthat the channel is a signaling protein that func-tions independently of cation influx and prior tocation binding of intracellular targets.

The use of La3+ as a Ca2+ substitute is con-strained by the narrow window of its cellularactivity, mainly owing to limited solubility inphysiological solutions. Also, the high affinityof La3+ for the Ca2+ binding site could affect thecross talk with atoms lining the channel pore,compromising the efficiency of the release sig-nal (∼10% of Ca2+ signal) (105). Therefore,the use of La3+ as a charge carrier to studyevoked release requires a highly sensitive secre-tory assay such as amperometry (76) or a strongsecretory signal such as insulin release in pan-creatic islets (106). Evoked release by a La3+-bound channel independent of cation influx in-spired a molecular approach to study secre-tion triggered by a Ca2+-impermeable channel(76).

Impermeable VGCC SupportsEvoked Secretion

The molecular strategy aimed at examiningwhether Ca2+ binding to the channel triggerssecretion independently of Ca2+ entry utilizeda channel mutant that binds Ca2+ at the channelpore but is unable to transport Ca2+ into thecell (Figure 2d ). The single-point mutationL775P at the sixth TM domain of segment II of

α11.2, the pore-forming subunit of the channel,abolishes the Ca2+ permeability of Cav1.2 (theL-type channel) (113) but spares the voltagesensitivity and Ca2+ binding at the pore (76). Asecond single-point mutation (T1066Y) con-ferring nifedipine resistance further mutatedthe Ca2+-impermeable mutant α11.2/L775P.This “permeate dead” nifedipine-resistantchannel was introduced into chromaffin cellsby the Semliki Forest virus. This mutationallows recordings of catecholamine secretionfrom the Ca2+-impermeable channel in thepresence of nifedipine when the secretionsignal mediated by the endogenous L-typechannels (85%) is blocked. Catecholaminesecretion mediated by the channel mutant orby a wild type (wt) nifedipine-resistant mutantintroduced into the cells as a control was simi-lar, suggesting that secretion is triggered priorto Ca2+ entry (76). The efficiency of evokedrelease mediated by the Ca2+-impermeablechannel was lower (∼ 25%) (76). This reduc-tion implies additional ancillary effects suchas elevated [Ca2+]i that could be essential foroptimizing the overall release process. Thisidea suggests an integrative mode of channelopening that initiates fusion-pore opening at<200 μs, followed by Ca2+ binding to syt1 andfusion-pore expansion at >1000 μs. However,foot amplitude, which relates directly to the sizeof the fusion pore and foot width and indicatesstability of the fusion pore (114, 115), remainsnearly identical in cells infected with the wtα11.2 or the Ca2+-impermeable α11.2/L775Psubunit (76). Also, Ca2+ entry has to enablesyt1 insertion in the membrane throughbinding to syt1 C2 domains. Independent ofhow close syt1 is to the membrane, C2 domaininsertion at the lipid bilayers could last seconds,whereas the release kinetics is faster (<200 μs).A more favorable interpretation is that the∼25% reduction in the release efficiency usingthe mutated channel could result from al-tered interaction(s) with Sx1A/SNAP-25/syt1compared with the control channel.

The data strongly support the view thatsecretion is triggered during Ca2+ binding atthe channel pore, independent of Ca2+ influx.

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This result is consistent with the excitosomemodel in which a change in channel conforma-tion during channel opening concomitant withCa2+ binding is sufficient to trigger secretionupstream of Ca2+ influx. In chromaffin cellsinfected with the Ca2+-impermeable channelL775P mutant, the increase in secretion of cat-echolamine by a selective Cav1.2 agonist, FPL64176, was independent of Ca2+ entry (108).The agonist-driven structural change affects se-cretion upstream of Ca2+ influx, consistent withthe role of the channel as a secretion-signalingmolecule (108).

MOLECULAR MECHANISM OFVGCC: RECONSTITUTION OFDEPOLARIZATION-EVOKEDTRANSMITTER RELEASE

An insightful understanding of the molecularmechanism of a signaling process such as vesi-cle fusion requires reconstitution of the releaseprocess in a cell-free system, as previouslydemonstrated for the β-adrenergic receptorthat mediates cyclic AMP elevation via the Gprotein–activated adenylyl cyclase (116). Theidentification of a limited set of proteins thatcontrols synaptic vesicle fusion significantlyincreased our understanding of the molecularmechanism of the release process (24). This setof proteins consists of SNARE proteins andaccessory proteins, Sec1/Munc18-related pro-teins, syt1, and complexins that regulate variousparts of the SNAREs’ activity (23, 33, 117, 118).

Reconstitution of neurotransmitter releasein earlier in vitro studies had fundamentallimitations. Initially, studies probed the ex-change of lipids between membranes and notthe release of synaptic-vesicle content. Often,only a subset of the key constituents was used,and most of the time the proteins were eithertruncated (e.g., lacking the TM domains) ormutated at the TM for better expression of therecombinant proteins. The time resolution ofphospholipid mixing induced by constant Ca2+

concentration was much too slow to accountfor the speed of secretion.

Later, a single-vesicle assay was introducedthat addressed the Ca2+-triggered burst of re-lease on a millisecond timescale by monitoringsimultaneous vesicle content and lipid mixing(30). Single-vesicle fusion demonstrated thatthe SNARE proteins by themselves were un-able to overcome the energy barrier and failedto induce fusion. The addition of syt1 to theassay was critical to achieve a full fusion be-tween the membranes and was enhanced bycomplexin (30). In this highly resolved assay,several cysteine residues of the SNAREs weremutated (e.g., C271S and C272S in Sx1A) (seeTransmission of the Signal from the Channelto the Exocytotic Machinery via Syntaxin 1A,below). Similar to previous reconstitution as-says, this high-resolution system could differen-tiate between single vesicle interaction, hemi-fusion, and a complete fusion. But this assay,like all other reconstituted systems, used Ca2+

as the trigger of secretion and not membranedepolarization, the physiological signal that in-volves channel opening and channel interac-tion(s) with the synaptic proteins.

A voltage-driven release was reconstitutedusing Xenopus oocytes coexpressing mammalianVGCC and the corresponding synaptic pro-teins (99). The reconstitution assay in oocytesmaintains the characteristic physiological traitsof neuronal exocytosis. Secretion is triggeredfrom a holding potential of −80 mV to 0 mV bytwo consecutive 500-ms pulses 100 ms apart inoocytes coexpressing Cav1.2, Cav2.1, or Cav2.3with Sx1A, SNAP-25, and syt1 (99). Vesicle fu-sion is monitored by changes in membrane ca-pacitance (Cm) according to Schmitt & Koepsell(119).

This assay uses full-length proteins that aretargeted to the correct location and operatewithin the native membrane of a living cell, asopposed to reconstitution assays that employtruncated recombinant proteins and artificialmembranes. Changes in capacitance are notdetected in oocytes that express SNAP-25and Sx1A without the channel or when thechannel is selectively blocked as well as innative oocytes. BotC1 and BotA, which respec-tively cleave Sx1A and SNAP-25, abolished



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secretion, demonstrating the essential roleof SNAP-25 and Sx1A in the reconstitutionof depolarization-induced exocytosis andconfirming the specificity of the assay (99). Incontrast, TetX cleavage of VAMP2 causes inhi-bition of the VAMP2-enhanced secretion, butdoes not block it (120). The partial inhibition byTetX excludes VAMP2 from the minimal set ofcore proteins essential for triggering secretion(99). The assay demonstrates that the minimal,essential core proteins for reconstituting se-cretion consist of the VGCC, Sx1A, SNAP-25,and syt1 (99). Other proteins that participatein various stages of the release process (e.g.,Munc18, Munc13, complexin, RIM, and oth-ers) should be tested for their role in shapingthe assembled protein core complex called theexcitosome complex. These proteins mightcontribute in many ways: VAMP2 to enhanceevoked release (99), RIM to tether Cav2.2 andCav2.1 to presynaptic active zones (36, 121–123), and cysteine string proteins as molecularchaperones in the assembly/disassembly ofexocytotic complexes with Cav2.1 (124).

The role of VAMP2 in evoked releaseas predicted by the reconstituted assay wasscrutinized in light of synaptobrevin knockoutresults (125). According to the excitosomemodel, Ca2+ binding at the pore of the channeltriggers fast secretion (1, 54). It depicts a fusionevent that is triggered subsequent to vesicledocking through the excitosome complex(syt1/channel/tSNAREs). This event occurssubsequent to SNAREs assembly, consistentwith Schoch et al.’s synaptobrevin/VAMPknockout mice analysis: “Full SNARE com-plexes are not required for fusion as such butcatalyze formation of transition states” (125).Schoch et al. (125) have demonstrated thatsucrose-triggered release, which releases alldocked vesicles, was ∼10-fold lower in thesynaptobrevin knockouts compared with con-trols, whereas the fast depolarization-triggeredrelease was ∼100-fold lower. These results areexpected according to the excitosme model.The nonreleasable/releasable docked vesiclesare in equilibrium, therefore a10-fold reductionin total docked vesicles would equally decrease

the releasable pool (Figure 1). Because theratio of nonreleasable/releasable varies up to10-fold or more, depolarization-evoked releaseshould drop ∼100-fold to a nearly undetectableresponse in synaptobrevin knockouts. Con-sistent with synaptobrevin not having a directeffect on the fusion machinery (99, 125), thesynaptobrevin knockout data can be explainedby a decrease in the production of docked vesi-cles, in good correlation with the excitosomemodel (1).

The reconstitution assay appears to faith-fully mimic evoked secretion. It is fast and ro-bust; it exhibits nonlinear Ca2+-concentrationdependence and is modulated by the type ofcharge carrier and the type of the VGCC (99).Nevertheless, the oocyte, which consists of thefrog’s homologous proteins and is clearly not anexcitable cell, is fundamentally different fromneurons. The seeming shortcomings are inher-ent to other ex vivo systems such as cortices ofsea urchin eggs, human embryonic kidney 293cells, and other in vitro systems. Compared withreconstitution assays that employ artificial lipidvesicles and purified proteins, the oocytes pro-vide a background that is neither completelyclean nor completely defined. Rather, oocyteslikely express multiple proteins that to some ex-tent could affect depolarization-induced exocy-tosis. However, the input of these proteins tothe secretion signal is nullified by the signif-icantly higher level of expression of the cor-responding heterologous proteins, making thecontribution of the endogenous proteins ratelimiting.

Reconstitution of depolarization-inducedexocytosis in oocytes offers significant advan-tages over other forms of reconstituted exocy-tosis. First, it uses the physiological trigger ofsynaptic exocytosis (membrane depolarization)under voltage-clamped conditions. Exocytosiscan be detected with more precision and shortertime resolution (subseconds), compared withminutes or hours in some other reconstitutionassays. Proteins expressed in Xenopus oocytesare full-length native proteins, which are prop-erly processed, targeted, and inserted at a livingcell membrane, as opposed to reconstitution

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assays that employ truncated and/or mutatedrecombinant proteins and utilize artificialmembranes. Second, reconstitution of secre-tion from oocytes offers exceptional controlover levels of expression of fully functionalchannels and full-length synaptic proteins withminimal contribution of the frog’s proteins.This experimental flexibility is instrumentalin rigorously testing the role of individualproteins in synaptic exocytosis and VGCCblends. Moreover, capacitance as a measureof exocytosis is simple, robust, and accurate(119).

HOW DOES THE VGCC TRIGGERNEUROTRANSMITTERRELEASE?

The results show that VGCCs most proba-bly, through a conformational change that cou-ples the voltage sensor domains and the chan-nel pore binding site, mediate fusion of dockedvesicles via a primed excitosome (see sidebar,Docked Vesicles According to the ExcitosomeModel). The question then arises as to howCa2+ binding at the channel confers the struc-tural change, how it is propagated, and whichof the exocytotic proteins is the target. Otherquestions related to the termination of the ex-ocytotic signal, spontaneous release, or facil-itation of release should be further explored,through examining the actual need of Ca2+ atthe channel pore independently of entry, andprior to binding to syt1 (see below).

The Channel Pore Triggers EvokedSecretion by Switching from theNonconductive to the ConductiveMode

Ca2+ binding at the selectivity filter of the chan-nel, the EEEE locus, is essential for voltage-driven transmitter release (Figure 2a,b). Atrest, Ca2+ occupies the high-affinity site of thepore (Kd < 1 μM) and the channel is in a low-energy state, compared with the high-energystate of the open channel. The probability ofthe channel undergoing a transition to the open

conformational state, which is a function of theenergy difference and the height of the energybarrier between the closed and open states, in-creases during membrane depolarization.

Channel opening exposes the low-affinity(Kd = 13.4 mM) Ca2+ binding site of thepore. Accommodation of two Ca2+ ions at thislow-affinity site converts the channel from anonconducting, high-affinity, single-ion site toan ion-conducting, low-affinity, multi-ion pore(101). The excitosome model proposes that thechange in the channel conformation during sat-uration of the low-affinity Ca2+ binding sitetriggers synchronous release independent ofsubsequent Ca2+ permeation (76, 105).

Hence, voltage perturbation signals syn-chronous release by converting a low-energy,single Ca2+ ion–bound closed channel toa high-energy, multi-Ca2+ion–bound openchannel. Cation interactions with atoms liningthe pore are predicted to induce significantstructural changes, as previously establishedin the K+ channel from Streptomyces lividans(K+-KcsA channel) (126). Thermodynamicmeasurements of ion binding at the K+-KcsAchannel showed that the channel’s ability toadopt a specific conductive structure stronglydepended on ion size, governed by the proteinstructure. The formation of the conductivestructure involves the selectivity filter atomsthat are in direct contact with bound ions, aswell as protein atoms associated with the pore,up to a distance of 15 A from the ion bindingsites (126). Analogously, synaptic proteinsfunctionally and physically associated with thechannel could detect changes, which propagatefarther away from the channel pore. Indeed, theSx1A/channel interaction is highly susceptibleto the type of the charge carrier residing in theselectivity filter (55, 75, 89, 107). These resultsillustrate the importance of conformationalcoupling between the voltage sensor domainand the channel pore domain, and confirm theputative contribution of cation interactionswith the atoms lining the channel pore tothe bidirectional intramembrane cross talkof channel/exocytotic machinery (see below)(101, 127).



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Transmission of the Signal from theChannel to the Exocytotic Machineryvia Syntaxin 1A

We predict that a signal initiated within theTM domain is propagated directly to meet itstarget within the membrane milieu. The TMdomain of Sx1A, which is the excitosome-onlyTM protein, is the most likely candidate tocapture a signal emanating from the channelopen pore. Functional interactions of the chan-nel with Sx1A involve two independent inter-faces, a cytosolic (15, 81, 85, 92) and a TM do-main (18, 74, 75, 88, 89, 128). The Sx1A cytoso-lic domain modifies channel activation throughinteraction with the cytosolic II-III loop of theα1 pore-forming subunit of the VGCC linkingTM6 of segment II and TM1 of segment III(88). The Sx1A TM domain negatively regu-lates current amplitude and affects evoked re-lease through interaction with a yet unknownsite at the channel (Figure 2e) (14, 16, 54, 74,75, 88, 128, 129).

Mutating two highly conserved vicinal cys-teine residues (C271V and C272V) of the Sx1ATM domain eliminates reconstituted evokedsecretion in oocytes (54, 99). Similarly, BotC1,which cleaves Sx1A, disrupts Sx1A interactionwith Cav1.2, Cav2.2, or Cv2.1 (54, 74, 99, 130,131, 132) and abolishes evoked secretion (28,120). Loss of the ability to modify channel ki-netics, which correlates with failure to triggerrelease (54, 75, 99, 129), stipulates a disruptionof the signaling interface between Sx1A and thechannel (54, 74, 75, 128).

These results are consistent with a model inwhich a signal originating at the channel andtransmitted to the Sx1A TM domain is lostby Sx1A TM mutants (74, 128) or by BotC1cleavage (129). Therefore, the Sx1A TM do-main emerges as a well-positioned interface torespond to conformational changes associatedwith the open pore of the channel. Further-more, Sx1A modulation of channel kinetics issusceptible to the type of cation residing at thechannel pore (55, 89). Analogous to the activa-tion of β-adrenergic receptors by structurallysimilar agonists (133), Ca2+, Sr2+, Ba2+, and

La3+, corresponding to the channel ligands, dif-ferently modify the channel interaction withSx1A and affect depolarization-evoked releaseby the engagement of distinct subsets of confor-mational changes in the channel pore (89). Thesusceptibility of the channel/Sx1A interface tothe type of cation occupying the pore pro-vides additional evidence for intramembranesignaling between these two proteins. Defini-tive characterization of intramembrane signal-ing and its role in exocytosis still needs to beconfirmed in a physiological secretory system.

THE CURRENT MODEL VERSUSTHE EXCITOSOME MODEL

The correlation between the overall Ca2+ affin-ity to syt1 and the Ca2+ sensitivity of neu-rotransmitter release led researchers to estab-lish syt1 as the Ca2+ sensor in synchronousrelease (see Could Synaptotagmin 1 Accountfor the Speed of the Release Process, above)(63). When introduced by homologous recom-bination into the endogenous synaptotagmin Igene in mice, the R233Q point mutation at thesyt1 C2A domain decreased the Ca2+ sensitiv-ity of neurotransmitter release twofold, did notchange the size of the readily releasable poolof vesicles, and did not change spontaneous re-lease (Figure 3) (but see 64). These results for-mulated the major basis to suggest syt1 as thecalcium sensor of secretion.

Three experimental data points analyzed ac-cording to the current model and the excito-some model demonstrate that syt1 functions asa vesicle-priming protein.

1. “Action potentials elicited by brief depo-larizations induce synaptic amplitudes consis-tently lower in the R233Q Syt1 mutant neu-rons compared to wt neurons” (63).Current model: The twofold decrease in theCa2+ sensitivity of neurotransmitter releaseresults from a decrease in the Ca2+ affinityof the R233Q syt1 mutant.Excitosome model: The twofold decrease inthe Ca2+ affinity of the R233Q syt1 mutant

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Nonreleasable vesiclessyt1 (–Ca2+)

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Figure 3The excitosome model explains syt1 knockin mutant results. (a) Mutating syt1 R233Q, the positively charged residue surrounding theCa2+ binding sites of the C2A domain, caused a twofold decrease in the overall Ca2+ affinity of syt1 and decreased the Ca2+ sensitivityof neurotransmitter release twofold as shown by inhibitory postsynaptic currents (IPSC) (inset) (63). According to the excitosomemodel, the reduction in Ca2+ affinity for syt1 decreases the number of primed excitosome complexes, consequently lowering theprobability of release. (b) The syt1 D232N knockin mutation increases evoked release by neutralizing the syt1 C2 Ca2+ binding sites(63), which, according to the excitosome model, primes more releasable vesicles. The increase in the fraction of the releasable poolleads to an increase in evoked secretion.

results from the decrease in the number ofprimed releasable vesicles (Figure 3; seesidebar, Docked Vesicles According to theExcitosome Model).2. “A decrease in spontaneous release inR233Q Syt1 mutant neurons is similar to therelative decrease in evoked release in R233Qmutant” (64).Current model: The twofold decrease inthe Ca2+ sensitivity of spontaneous releaseresults from a decrease in the Ca2+ affinity ofthe R233Q syt1 mutant and establishes syt1as the Ca2+ sensor of spontaneous release.Excitosome model: Spontaneous release re-sults from the spontaneous fusion of channel-tethered primed vesicles during the sponta-

neous opening of a channel (see SpontaneousRelease as Viewed by the Current Model andthe Excitosome Model, below). The lowernumber of primed excitosome complexes gen-erated by the twofold decrease in Ca2+ affinityto syt1 lowers the number of releasable vesi-cles, consequently decreasing spontaneous re-lease the way it decreases synchronous release.3. “The R233Q mutant does not alter the sizeof the readily releasable pool of neurotrans-mitters” (63).Current model: Hypertonic sucrose (0.5 M)releases all docked vesicles in a Ca2+-independent mechanism. Because the size ofdocked vesicles released by sucrose is not al-tered in the R233Q mutant, it substantiates



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the view that syt1 is the Ca2+ sensor of theevoked release.Excitosome model: R233Q mutation in-creases the proportion of releasable to nonre-leasable pools in the terminal without affectingthe total number of docked vesicles. There-fore, hypertonic sucrose, which releases theentire pool of docked vesicles, should not beaffected by the R233Q mutation.

According to the excitosome model, themost plausible explanation for the impairedvesicular release probability in syt1 R233Q orthe increase in vesicular release in syt1 R2328Nknockin neurons (22, 63, 64) is a changein the ratio of primed/nonprimed vesicles(Figure 3). syt1 controls the ratio of the twopools of docked vesicles and thus functions asa vesicle-priming protein rather than a submil-lisecond fusion-triggering protein.

SPONTANEOUS RELEASE ASVIEWED BY THE CURRENTMODEL AND THE EXCITOSOMEMODEL

Fatt & Katz (134) suggested that the sponta-neous fusion of vesicles represents backgroundactivity of neurons triggered by spontaneousCa2+ fluctuations. Spontaneous release inindividual excitatory synapses of the pyramidalneurons in the CA1 region of the hippocampusis a rare event and occurs only once every 2–3 hand once every 3 min at the inhibitory synapses.Spontaneous fusion events obtained in theabsence of presynaptic potential are calledminiature current (minis) and thought to reflectrelease of single vesicles (134). In PC12 cells aswell as in dendrites, they occur repetitively atthe same locations, frequently at branch points,suggesting that event sites are related to selec-tive molecule-oriented sites (135). Recently,Klingauf and colleagues (136) presented datathat favor a common origin of synaptic vesiclesundergoing spontaneous and evoked fusion,whereas others concluded that distinct synapticvesicle populations participate in evokedrelease and spontaneous release (137, 138,

139). The Ca2+ dependency of spontaneousrelease and its potential physiological role ledto a search for the Ca2+ sensor of the process.

Spontaneous Release Accordingto the Current Model

Investigators proposed that two Ca2+ sensorsare responsible for triggering spontaneous re-lease, the double C2 domain family of proteinsthat could account for approximately half ofthe spontaneous release events in hippocampalneurons (140), or syt1 (64). Spontaneous releasewas lower in neurons cultured from knockinR233Q syt1 neurons, similar to the decreasein action potential–driven exocytosis (63; alsosee Reference 64). These results were takenas evidence that, as for synchronous release,syt1 is also the Ca2+-dependent sensor forspontaneous release (see above) (22, 63, 141).

Spontaneous Release Accordingto the Excitosome Model

Fatt & Katz (142) suggested that “Sponta-neous excitation might simply be the resultof excessive voltage noise across the nervemembrane,” where “the ‘noise’ voltage acrossthe axon membrane may become so large,at a sufficiently minute structure, that it mayoccasionally exceed the threshold level at somepoint. By ‘noise’ voltage is meant the randomfluctuation of the resting potential due tothermal agitations within the membrane.”The excitosome model, consistent with Fatt& Katz’s view, suggests that spontaneousrelease is driven by random fluctuations of theresting potentials that coincide with stochasticopening of the Ca2+ channel at rest, triggeringfusion of channel-tethered vesicles.

Spontaneous release triggered by the activa-tion of mechanosensitive or other channels alsocoincides with VGCC opening (143). These re-sults are consistent with voltage perturbationsthat eventually open the VGCC and triggerspontaneous release in a mechanism similarto evoked release. Similarly, nicotine binding

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to presynaptic acetylcholine receptors inducesspontaneous release of glutamate onto layerV pyramidal neurons of the prefrontal cortex.Because nicotine opens Na+/K+ channelsand depolarizes the presynaptic membrane,the nicotine-induced release involves VGCCopenings that trigger transmitter release (144,145). Therefore, a reduction in the Ca2+ affinityof syt1 in syt1 knockin mutant neurons, whichdecreases the releasable pool, would affectnicotine-induced secretion the way it affectsdepolarization-evoked release (22, 64). It is thesame releasable pool located at the same sitethat mediates both the synchronous and spon-taneous release (134, 137, 146). The excitosomemodel predicts that synchronous, spontaneous,and nicotine-induced release involve the sameexcitosome-primed vesicles positioned withinthe active zone. Furthermore, because Ca2+

occupancy of syt1 regulates the ratio of re-leasable/nonreleasable vesicles, all three modesof secretion should be equally modified by thecorresponding syt1 mutations (see above).

Moreover, elevating [Ca2+]i by thapsigarginor caffeine through Ca2+ release from internalstores would expand the fraction of releasablevesicles upon binding to syt1, increasing theprobability of secretion. Any increase in theratio of releasable/nonreleasable vesicles atthe terminal, either through elevating [Ca2+]i

internally or from the extracellular medium,would increase synchronous release by increas-ing the probability of vesicle fusion from alarger pool (see sidebar, Facilitation of Secre-tion as Explained by the Excitosome Model).Thus, the VGCC appears to meet the criteriafor a Ca2+ sensor protein of spontaneous re-lease, whereas syt1 emerges as a Ca2+-bindingprotein that primes vesicles for fusion eitherspontaneously or through an action potential.

WHAT MAKES THE VGCCAN ATTRACTIVE CALCIUMSENSOR OF SECRETION?

1. The presence of both voltage-sensingand Ca2+-sensing determinants within asingle molecule provides an ultimate

FACILITATION OF SECRETION ASEXPLAINED BY THE EXCITOSOME MODEL

Facilitation is a Ca2+-dependent elevation of the probability oftransmitter release during the repetitive stimulation of synapses.It is triggered during brief trains of action potentials and causessuccessive postsynaptic potentials (147). Researchers have ob-served this form of synaptic enhancement at neuromuscular andinvertebrate synapses (148) and in the mammalian CNS (149).

The elevated [Ca2+]i remaining from previous stimuli, termedresidual [Ca2+]i, causes facilitation by increasing the probabil-ity of transmitter release (147). However, a simple linear sum-mation cannot explain facilitation. Transmitter release rates in-duced by presynaptic action potentials were compatible with briefmicrodomain [Ca2+]i transients with peak amplitudes of 10 μM(150) or 25 μM (151), implying that facilitation results from an in-creased microdomain [Ca2+]i signal for transmitter release. Thisview was further supported by evidence that neither the Ca2+

cooperativity nor the Ca2+ dependence of release kinetics fortransmitter release changes during facilitation.

Short-Term Synaptic Facilitation as Explained by theExcitosome Model

A train of action potentials leads to a higher [Ca2+]i comparedwith a single action potential, consequently priming a largernumber of vesicles through Ca2+ binding to syt1. Because theprobability of secretion from a larger pool of releasable vesiclesis higher, the most straightforward explanation for facilitationwould be an increase in the probability of vesicle fusion froma larger fraction of releasable vesicles (Figure 1). The sugges-tion that facilitation is proportional to the occupancy of syt1 isconsistent with a previous proposition for the involvement of ahigh-affinity Ca2+ binding site of a putative Ca2+-binding protein(152, 153).

control of the fast evoked-release process.Voltage-driven conformational change isessential for exposing the Ca2+ bindingsite at the pore of the channel, enablingCa2+ binding at the low affinity site ofthe channel. The signal generated at thechannel pore is propagated directly toSx1A via intramembrane signaling.

2. Docked vesicles are tethered to the mem-brane by the excitosome complex, which



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Facilitation: aCa2+-dependentelevation of theprobability oftransmitter releaseduring repetitivestimulation of synapses

consists of the channel, Sx1A, SNAP-25,and syt1. Accordingly, all docked vesiclesare tethered to the channel. Docked vesi-cles are classified into two pools by theCa2+ occupancy of syt1 C2 domains: a re-leasable pool consisting of vesicles associ-ated with a primed excitosome, in whichsyt1 is Ca2+ bound, and a nonreleasablepool consisting of vesicles associated witha nonprimed excitosome, in which syt1 isCa2+ free. The two pools of docked vesi-cles are in a dynamic equilibrium.

3. Vesicles tethered through a primed ex-citosome (releasable) are signaled to fusewith the membrane by a conformationalchange during Ca2+ binding at the poreof the channel. The assembled primed ex-citosome enables a submillisecond rate ofsynaptic transmission (Figure 4).

4. BAPTA- and EGTA-treated cells chelatefree Ca2+ and thereby diminish vesi-cle priming. By preventing Ca2+ bind-ing to syt1 and the priming of nonre-leasable to releasable vesicles, BAPTAand EGTA eventually block the secre-tory signal. However, neither BAPTAnor EGTA inhibits fast secretion fromvesicles in the releasable pool in whichsyt1 within the excitosome is Ca2+ bound.

5. Ca2+ elevation by an action potentialthrough binding to syt C2 domainsinserts syt1 into the membrane andpromotes new interactions with thet-SNAREs and the channel. By minimiz-ing the activation energy, this primingstep converts the nonreleasable to re-leasable vesicles, ready for fusion. syt1,a vesicle-priming protein, regulates thenumber of releasable vesicles, controllingthe robustness of secretion.

6. The increase in the releasable dockedvesicles, normally at equilibrium withdocked nonreleasable vesicles at thesynaptic cell, determines the physio-logical transient increase in transmitterrelease probability during repeatedstimulation at short time intervals (i.e.,facilitation).

CONCLUDING REMARKS

This chapter addressed mainly the ability oftwo calcium-binding proteins, VGCC andsyt1, to perform as Ca2+ sensors of synaptictransmission. The emerging view is that theVGCC acts as a dynamic and regulatorymolecular switch that triggers transmitterrelease, with the speed of conformationalchange at ∼100–1000 μs. During an actionpotential, the channel shuttles from a closed toan open state through conformational changescoupled to the channel pore. The conversion ofa closed single-ion pore to an open Ca2+-boundmulti-ion pore channel switches on synaptictransmission. Similarly, voltage-driven Ca2+

binding at the L-type Ca2+ channel triggersexcitation-contraction coupling prior to Ca2+

influx in neonate cardiomyocytes (154). Hence,analogous to an agonist activating a receptor,cation binding at the open pore of the channel(during membrane depolarization) appears tobe a general mechanism by which the VGCCtransmits a signal into the cell. Transmitterrelease is switched off when the channel re-sumes a closed single-ion pore conformation.The vesicular protein, syt1, emerges as acrucial vesicle-priming protein that maintainsand coordinates the balance of releasableand nonreleasable vesicles at the terminal,resolving the robustness of the secretorysignal.

The studies also highlight a putative in-tramembrane signaling mechanism in whichfunctionally associated membrane proteins(VGGG and Sx1A) interact with each otherwithin the membrane bilayer. Intramembranesignaling enables rapid transduction of aconformational change between two TMproteins, essential for triggering fast secretion.The role of the VGCC as a signaling moleculeis twofold: Initially, it transduces a signal to theexocytotic machinery to stimulate secretion ofreleasable vesicles, and subsequently, it intro-duces Ca2+ into the cell providing syt1 withCa2+, which is required for priming vesiclestoward a new cycle of fast secretion. The exactmolecular rearrangements obligatory for the

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Primedexcitosome

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Figure 4The sequence of events of depolarization-evoked secretion. (a) At any given time, two types of dockedvesicles are tethered to the membrane through the excitosome complex. When syt1 is Ca2+ bound, thevesicle is releasable (primed), and when syt1 is Ca2+ free, the vesicle is nonreleasable (unprimed). (b) Uponarrival of an action potential, the releasable vesicle fuses with the membrane within 100–1,000 μs. Fusion istriggered in response to a confomational change initiated at the pore of the channel and propagated throughintramembrane signaling to the exocytotic machinery via Sx1A. Concomitantly, Ca2+ that permeatedthrough the channel into the cell during channel stimulation bind to syt1, priming the nonreleasable toreleasable vesicles, making them ready to fuse with the membrane by the next incoming action potential.



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opening and closing of the fusion pore throughwhich the transmitter escapes from the vesicleto the extracellular medium and the role ofthe VGCC in the fusion pore await further

investigation. Future studies, such as those thatunravel the 3D structure of the VGCC, wouldhelp in elucidating the precise mechanism ofsynaptic transmission.

SUMMARY POINTS

1. The VGCC functions as a Ca2+ binding signaling protein.

2. The VGCC operates as the Ca2+ sensor of synchronous and spontaneous secretion.

3. syt1 operates as the Ca2+ sensor of vesicle priming.

4. A voltage-driven conformational change enables Ca2+ occupancy at the selectivity filter.This conformational coupling between the voltage-sensing domain and Ca2+ binding siteat the pore is propagated directly via the TM domains, signaling the exocytotic proteinto trigger vesicle fusion.

5. Intramembrane signaling between the VGCC and the TM domain of Sx1A appears tomediate the excitation-secretion coupling.

6. The conversion of the selectivity filter from a high nonconducting state to a low-affinityconducting state is the switch that turns on vesicle fusion.

7. The return of the channel to the nonconducting state stops the signal and terminatessecretion.

8. Two different pools of docked vesicles are tethered to the membrane via the excitosomecomplex, which consists of the channel, Sx1A, SNAP-25, and syt1. The nonreleasablevesicles are tethered to the membrane via a nonprimed excitosome complex in whichsyt1 is Ca2+ free. The releasable vesicles are tethered to the membrane via a primedexcitosome complex in which syt1 is Ca2+ bound.

FUTURE ISSUES

1. What is the detailed architecture of the excitosome complex at sites of exocytosis? Howdoes that architecture change during Ca2+ binding to syt1?

2. How general is the mechanism in which a signal can be propagated directly from a cellmembrane channel to an intracellular protein?

3. How general is the intramembrane signaling mediated by TM domains of two differentproteins?

4. How does kinetics of different types of VGCC affect vesicle fusion?

5. Does excitation-contraction coupling in the heart share a common molecular mechanismwith the excitosome model?

6. Is the VGCC part of fusion pore?

7. Does the coupling of different VGCCs with the exocytotic proteins determine the outputof a synapse?

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8. Does the strength of certain synapses correlate with the inherent distribution of releasableto nonreleasable vesicles?

DISCLOSURE STATEMENT

The author is not aware of any affiliations, memberships, funding, or financial holdings that mightbe perceived as affecting the objectivity of this review.

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The Voltage-Gated Calcium Channel Functions as the Molecular Switch of Synaptic Transmission

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