In the exocytosis of neurotransmitter, fusion pore opening represents the first instant of fluid contact between the vesicle lumen and extracellular space. The existence of the fusion pore has been established by electrical measurements, but its molecular composition is unknown. The possibility that synaptotagmin regulates fusion pores was investigated with amperometry to monitor exocytosis of single dense-core vesicles. Overexpression of synaptotagmin I prolonged the time from fusion pore opening to dilation, whereas synaptotagmin IV shortened this time. Both synaptotagmin isoforms reduced norepinephrine flux through open fusion pores. Thus, synaptotagmin interacts with fusion pores, possibly by associating with a core complex of membrane proteins and/or lipid.

CAPS-1 is required for Ca2+-triggered fusion of dense-core vesicles with the plasma membrane, but its site of action and mechanism are unknown. We analyzed the kinetics of Ca2+-triggered exocytosis reconstituted in permeable PC12 cells. CAPS-1 increased the initial rate of Ca2+-triggered vesicle exocytosis by acting at a rate-limiting, Ca2+-dependent prefusion step. CAPS-1 activity depended upon prior ATP-dependent priming during which PIP2 synthesis occurs. CAPS-1 activity and binding to the plasma membrane depended upon PIP2. Ca2+ was ineffective in triggering vesicle fusion in the absence of CAPS-1 but instead promoted desensitization to CAPS-1 resulting from decreased plasma membrane PIP2. We conclude that CAPS-1 functions following ATP-dependent priming as a PIP2 binding protein to enhance Ca2+-dependent DCV exocytosis. Essential prefusion steps in dense-core vesicle exocytosis involve sequential ATP-dependent synthesis of PIP2 and the subsequent PIP2-dependent action of CAPS-1. Regulation of PIP2 levels and CAPS-1 activity would control the secretion of neuropeptides and monoaminergic transmitters.

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca2+ ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca2+-sensing module. Here, we report that Ca2+ drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca2+ are mediated by a set of conserved acidic Ca2+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca2+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca 2؉ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a cytoplasmic domain largely comprised of two C2 domains designated C2A and C2B. We have determined how deep the Ca 2؉ -binding loops of Ca 2؉ ⅐C2A penetrate into the lipid bilayer and report mutations in synaptotagmin that can uncouple membrane penetration from Ca 2؉ -triggered interactions with the SNARE complex. To determine whether C2A penetrates into the vesicle ("cis") or plasma ("trans") membrane, we reconstituted a fragment of synaptotagmin that includes the membrane-spanning and C2A domain (C2A-TMR) into proteoliposomes. Kinetics experiments revealed that cis interactions are rapid (<500 s). Binding in the trans mode was distinguished by the slow diffusion of trans target vesicles. Both modes of binding were observed, indicating that the linker between the membrane anchor and C2A domain functions as a flexible tether. C2A-TMR assembled into oligomers via a novel N-terminal oligomerization domain suggesting that synaptotagmin may form clusters on the surface of synaptic vesicles. This novel mode of clustering may allow for rapid Ca 2؉ -triggered oligomerization of the protein via the membrane distal C2B domain. Ca2ϩ -triggered fusion of synaptic vesicles with presynaptic plasma membrane mediates the release of neurotransmitters from neurons. The release process is extremely fast, occurring on the sub-millisecond time scale (1-3). Synaptotagmin I (hereafter referred to as synaptotagmin) is a Ca 2ϩ -binding synaptic vesicle protein that has been proposed to function as a Ca 2ϩ sensor that triggers release in response to Ca 2ϩ influx (4 -6). Structurally, synaptotagmin spans the vesicle membrane once and has a short intravesicular N-terminal domain and a large C-terminal cytoplasmic region that contains two C2 domains, designated C2A and C2B 1 (7). A charged "linker" segment connects the C2 domains to the transmembrane domain.The structure and biochemical properties of the C2A domain have been studied in detail. This domain forms a compact eight-stranded ␤-sandwich structure. Three flexible loops that protrude from one end of the domain mediate the binding of two to three Ca 2ϩ ions (8 -10). Recent fluorescence and NMR studies demonstrated that Ca 2ϩ -binding loops 1 and 3 penetrate into lipid bilayers in response to binding Ca 2ϩ (11)(12)(13). The second C2 domain (C2B) mediates the Ca 2ϩ -dependent oligomerization of synaptotagmin (14, 15), potentially clustering the cytoplasmic domain into a collar or ring-like structure that may regulate the opening or dilation of the fusion pore (16). Both C2 domains cooperate to mediate high affinity Ca 2ϩ -dependent binding of synaptotagmin to the plasma membrane proteins syntaxin (12,14) and 18). Syntaxin and SNAP-25 form a high affinity ternary complex with the synaptic vesicle protein synaptobrevin (19,20). This heterotrimer, designated the SNARE complex, has been proposed to function as the cor...

Real-time voltammetry measurements from cracked PC12 cells were used to analyze the role of synaptotagmin–SNARE interactions during Ca2+-triggered exocytosis. The isolated C2A domain of synaptotagmin I neither binds SNAREs nor inhibits norepinephrine secretion. In contrast, two C2 domains in tandem (either C2A-C2B or C2A-C2A) bind strongly to SNAREs, displace native synaptotagmin from SNARE complexes, and rapidly inhibit exocytosis. The tandem C2 domains of synaptotagmin cooperate via a novel mechanism in which the disruptive effects of Ca2+ ligand mutations in one C2 domain can be partially alleviated by the presence of an adjacent C2 domain. Complete disruption of Ca2+-triggered membrane and target membrane SNARE interactions required simultaneous neutralization of Ca2+ ligands in both C2 domains of the protein. We conclude that synaptotagmin–SNARE interactions regulate membrane fusion and that cooperation between synaptotagmin's C2 domains is crucial to its function.

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Assembly of the plasma membrane proteins syntaxin 1A and SNAP-25 with the vesicle protein synaptobrevin is a critical step in neuronal exocytosis. Syntaxin is anchored to the inner face of presynaptic plasma membrane via a single C-terminal membrane-spanning domain. Here we report that this transmembrane domain plays a critical role in a wide range of syntaxin proteinprotein interactions. Truncations or deletions of the membrane-spanning domain reduce synaptotagmin, ␣/␤-SNAP, and synaptobrevin binding. In contrast, deletion of the transmembrane domain potentiates SNAP-25 and rbSec1A/nsec-1/munc18 binding. Normal partner protein binding activity of the isolated cytoplasmic domain could be "rescued" by fusion to the transmembrane segments of synaptobrevin and to a lesser extent, synaptotagmin. However, efficient rescue was not achieved by replacing deleted transmembrane segments with corresponding lengths of other hydrophobic amino acids. Mutations reported to diminish the dimerization of the transmembrane domain of syntaxin did not impair the interaction of full-length syntaxin with other proteins. Finally, we observed that membrane insertion and wild-type interactions with interacting proteins are not correlated. We conclude that the transmembrane domain, via a lengthdependent and sequence-specific mechanism, affects the ability of the cytoplasmic domain to engage other proteins.Syntaxin 1A was initially identified as a 35 kDa protein in the plasma membrane of amacrine cells (1), as a subunit of Ca 2ϩ channels (2, 3) and as a synaptotagmin-binding protein (4). Since these initial reports, the function of syntaxin as a central component in the synaptic vesicle membrane fusion machinery has been well established (reviewed in Refs. 5-7). Syntaxin forms a putative membrane fusion apparatus by assembling into a fourhelix bundle (8) with the plasma membrane protein SNAP-25 1 and the synaptic vesicle protein synaptobrevin, to form a SNARE complex (9). Assembly of this complex is necessary (10, 11) and may be sufficient to drive membrane fusion (12)(13)(14). One current view is that the zippering together of the four-helix bundle drives membrane fusion by pulling the vesicle and target membranes together (8,12,15). In this model, the transmembrane domains (TMDs) of synaptobrevin and syntaxin would form part of a fusion pore (16). Thus, structure-function relationships of these TMDs may reveal insights into the mechanism of membrane fusion (13,14,(17)(18)(19).Syntaxin functions as a key element in membrane traffic and membrane fusion by interacting with a wide range of other proteins. The many binding partners of syntaxin, in excess of twenty, include rbSec1A/nsec-1/munc18 (20 -22), CSP (23), syntaphilin (24), ␣/␤-SNAP (9, 25), sec6/8 (26), tomosyn (27), Munc-13 (28), and as mentioned above synaptotagmin (4, 29) as well as a growing assortment of channels/receptors (see, for example Refs. 2, 3, 30 -32).Biochemical studies of syntaxin, including structural determinations (8, 33, 34), have made almost exclusive use of the ...

Rotating disk electrode voltammetry was used to measure the time-resolved inward transport of dopamine into human embryonic kidney cells expressing the human transporter for dopamine and a kinetic mechanism of transport is hypothesized. Dopamine transport in this preparation was highly concentrative, with a 10(6)-10(7) inward bias, first order in dopamine and the K(m) and V(max) were found to be 1.6 microM and 18 pmol/sec x 10(6) cells), respectively. The hDAT turnover was estimated to be approximately 18 s(-1) and the second order rate constant of association of dopamine with hDAT was approximately 10(7) M(-1)s(-1). Dopamine transport was found to have a second order dependence on Na(+) (K(Na) approximately 100 mM) and a first order dependence on Cl(-) (K(Cl) approximately 12 mM). Multisubstrate analyses suggested that hDAT operates with an ordered kinetic mechanism in which Na(+) binds first to the transporter protein, dopamine second, and Cl(-) last before translocation of dopamine into or across the membrane. Cocaine competitively inhibited dopamine transport (reaction order of unity and K(i) approximately 0.34 microM) with no discernible effect at the Na(+) and Cl(-) binding sites. These results differ from those of previous studies conducted in preparations of the striatum and nucleus accumbens. Comparisons of the variant results are made and an analysis of the differing apparent kinetic mechanisms is presented.