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Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C terminus depending on their energy of transfer from water to the membrane interface, following a Boltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets.chromaffin cell | patch clamp capacitance measurement | caged calcium | amperometry | electrochemical detector array T he SNARE proteins (1) mediate release of stored secretory products by exocytosis. In neurosecretion, the t-SNAREs syntaxin and SNAP-25 in the plasma membrane bind the v-SNARE synaptobrevin II (sybII, also known as VAMP2), which is anchored to the vesicle membrane by a single transmembrane (TM) domain. Upon stimulation, the SNARE complex is thought to zip up more tightly proceeding in a vectorial manner from the N to the C terminus, toward the TM domains of sybII and syntaxin (2-5), thereby transferring a force to the membranes (6). However, the molecular mechanism by which this force leads to opening of the fusion pore has not been determined (7). Several models have been proposed to explain the mechanism of fusion pore formation. In the lipid-stalk-hemifusion hypothesis, the outer and the inner leaflets of the two membranes merge via formation of a hemifusion intermediate in response to forces exerted by proteins surrounding the fusion site (8). In an alternative proteinaceous fusion pore model, the fusion pore is lined by the TM domain of syntaxin (9) and possibly synaptobrevin (10). However, it is not immediately evident how the hydrophobic transmembrane domains can line an aqueous fusion pore that allows for ion permeation by electrodiffusion (11). When the C-terminal SNARE domain interactions are reduced by mutating or deleting the C terminus of SNAP-25, or when flexible linkers are introduced between the sybII TM domain and its SNARE domain, the rate of exocytosis is reduced (12-16) and the flux of transmitter through the early fusion pore is decreased (16,17), consistent with a structural change in the fusion pore. In an attempt to interpret these findings, a proteolipidic fusion pore model has been proposed, in which the fusion pore is formed by a molecular complex of both lipids and SNARE proteins (17). However, even this model does not explain the molecular mechanism by which the N-to C-terminal zipp...

The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.

Formation of a fusion pore between a vesicle and its target membrane is thought to involve the so-called SNARE protein complex. However, there is no mechanistic model explaining how the fusion pore is opened by conformational changes in the SNARE complex. It has been suggested that C-terminal zipping triggers fusion pore opening. A SNAP-25 mutant named SNAP-25⌬9 (lacking the last nine C-terminal residues) should lead to a less-tight C-terminal zipping. Single exocytotic events in chromaffin cells expressing this mutant were characterized by carbon fiber amperometry and cell-attached patch capacitance measurements. Cells expressing SNAP-25⌬9 displayed smaller amperometric ''foot-current'' currents, reduced fusion pore conductances, and lower fusion pore expansion rates. We propose that SNARE/lipid complexes form proteolipid fusion pores. Fusion pores involving the SNAP-25⌬9 mutant will be less tightly zipped and may lead to a longer fusion pore structure, consistent with the observed decrease of fusion pore conductance.amperometry ͉ capacitance measurement ͉ chromaffin cell ͉ exocytosis ͉ patch clamp

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