Purified dihydropyridine-sensitive calcium channels from rabbit transverse-tubule membranes consist of three noncovalently associated classes of subunits: a (167 kDa), p (54 kDa), and y (30 kDa). Cleavage of disulfide bonds reveals two distinct a polypeptides and an additional component, 6.The a, subunit, a 175-kDa polypeptide that is not N-glycosylated, contains the dihydropyridine binding site, cAMP-dependent protein kinase phosphorylation site(s), and substantial hydrophobic domain(s). a2, a 143-kDa glycoprotein, has none of the properties characteristic of al but binds lectins and contains about 25% N-linked carbohydrate. a2 is disulfidelinked to 6, a 24-to 27-kDa glycopeptide. .3 (54 kDa) contains a cAMP-dependent phosphorylation site but is not N-glycosylated and does not have a hydrophobic domain. y (30 kDa) has a carbohydrate content of about 30% and extensive hydrophobic domain(s). Precipitation with affinity-purifiled anti-a, antibodies or a2-specific lentil lectin-agarose demonstrated that aja213yS behaves as a complex in the presence of digitonin or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, whereas the a26 complex dissociates from al.fy in the presence of Triton X-100. A model for subunit interaction and membrane insertion is proposed on the basis of these observations.

Almost all known intracellular fusion reactions are driven by formation of trans-SNARE complexes through pairing of vesicleassociated v-SNAREs with complementary t-SNAREs on target membranes. However, the number of SNARE complexes required for fusion is unknown, and there is controversy about whether additional proteins are required to explain the fast fusion which can occur in cells. Here we show that single vesicles containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin fuse rapidly with planar, supported bilayers containing the synaptic/exocytic t-SNAREs syntaxin-SNAP25. Fusion rates decreased dramatically when the number of externally oriented v-SNAREs per vesicle was reduced below 5-10, directly establishing this as the minimum number required for rapid fusion. Docking-to-fusion delay time distributions were consistent with a requirement that 5-11 t-SNAREs be recruited to achieve fusion, closely matching the v-SNARE requirement.lipid bilayer | membrane fusion | SNARE mechanisms | supported bilayer T rafficking of proteins in the cell-as well as secretion of physiological mediators such as hormones and neurotransmitters-depends on intracellular membrane fusion. With few exceptions, intracellular fusion reactions are driven by pairing of vesicle-associated v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with cognate t-SNAREs on the target membrane, resulting in a four-helix bundle (SNAREpin) that brings bilayers into close proximity (1-3). In cells, the action of SNAREs is regulated by auxiliary proteins, some of which, such as the members of the Sec1/Munc18-like (SM) family, are universally required components of the eukaryotic fusion machinery (4). Whether SNAREs alone can catalyze fusion at physiologically meaningful rates in the absence of modulating proteins or peptides (5-9) remains controversial. In addition, it is unknown how many SNAREpins are required to produce fusion. Here, using an in vitro assay that can resolve single docking and fusion events, we show 5-10 SNAREpins mediate fast fusion in the absence of any auxiliary proteins.Reconstituted fusion assays have played a key role in elucidating mechanisms of SNARE-mediated membrane fusion (1, 2, 5, 6, 10, 11). SNARE proteins reconstituted into small unilamellar vesicles (SUVs) fused bilayers in a bulk fusion assay, albeit with slow kinetics (1, 2). More recently, single SUVs containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin were shown to fuse rapidly with planar, supported bilayers (SBLs) containing the synaptic/exocytic t-SNAREs syntaxin 1-SNAP25, with single fusion events occurring in ∼10-100 ms (7, 9) to seconds (8, 12). However, the SNAP25 subunit of the t-SNARE was not required (8, 9), or an artificial peptide was needed (7), raising questions about the physiological relevance of these results. These, and other studies of SNARE-mediated membrane fusion, used lipid bilayers where the active fusion catalysts were the only proteins present. By contrast, natural intracellular membranes are popula...

Neurotransmitter release involves the assembly of a heterotrimeric SNARE complex composed of the vesicle protein synaptobrevin (VAMP 2) and two plasma membrane partners, syntaxin 1 and SNAP-25. Calcium in¯ux is thought to control this process via Ca 2+ -binding proteins that associate with components of the SNARE complex. Ca 2+ /calmodulin or phospholipids bind in a mutually exclusive fashion to a C-terminal domain of VAMP (VAMP 77±90 ), and residues involved were identi®ed by plasmon resonance spectroscopy. Microinjection of wild-type VAMP 77±90 , but not mutant peptides, inhibited catecholamine release from chromaf®n cells monitored by carbon ®bre amperometry. Pre-incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibited Ca 2+ -dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppressed secretion. In the presence of TeNT, exocytosis was restored by transfection of TeNT-resistant (Q 76 V, F 77 W) VAMP, but additional targeted mutations in VAMP 77±90 abolished its ability to rescue release. The calmodulin-and phospholipid-binding domain of VAMP 2 is thus required for Ca 2+ -dependent exocytosis, possibly to regulate SNARE complex assembly. Keywords: neuroendocrine cells/secretory vesicle/ SNARE/tetanus toxin IntroductionNeurones and neuroendocrine cells release transmitters and neuropeptides by calcium-dependent exocytosis of the contents of vesicles docked at the plasma membrane. This process requires assembly of trimeric SNARE complexes formed by the vesicle-associated membrane protein synaptobrevin (VAMP 2) and two partners that are expressed mainly in the plasma membrane, syntaxin 1 and synaptosome-associated protein of 25 kDa (SNAP-25) (reviewed by Jahn and Sudhof, 1999;Lin and Scheller, 2000;Mayer, 2001). Analysis of a minimal complex composed uniquely of the interacting domains of these three proteins has revealed a parallel bundle of four a-helices (one from VAMP 2, one from syntaxin 1 and two from SNAP-25) twisted into a superhelical structure (Sutton et al., 1998). Extrapolation of these data to a situation in which VAMP 2 and syntaxin 1/SNAP-25 are anchored in distinct lipid bilayers (i.e. docked vesicle membranes and plasma membranes, respectively) led to a proposal for trans SNARE complex function. The zippingup of SNARE complexes from the N-terminus to the C-terminus would pull the opposing C-terminal transmembrane anchors towards each other and promote membrane fusion at the vesicle±plasma membrane interface.Abundant evidence from the use of botulinum and tetanus toxins (BoNT and TeNT, respectively), which inhibit transmitter release by cleaving SNARE proteins (Xu et al., 1998;Hua and Charlton, 1999), as well as mutagenesis in invertebrates and mice (Fergestad et al., 2001;Schoch et al., 2001;Washbourne et al., 2002), have consolidated the view that SNARE proteins are required for exocytosis. However, the precise role of SNARE complex assembly in membr...

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Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.

Autosomal dominant epilepsy with auditory features results from mutations in leucine-rich glioma-inactivated 1 (LGI1), a soluble glycoprotein secreted by neurons. Animal models of LGI1 depletion display spontaneous seizures, however, the function of LGI1 and the mechanisms by which deficiency leads to epilepsy are unknown. We investigated the effects of pure recombinant LGI1 and genetic depletion on intrinsic excitability, in the absence of synaptic input, in hippocampal CA3 neurons, a classical focus for epileptogenesis. Our data indicate that LGI1 is expressed at the axonal initial segment and regulates action potential firing by setting the density of the axonal Kv1.1 channels that underlie dendrotoxin-sensitive D-type potassium current.LGI1 deficiency incurs a >50% down-regulation of the expression of Kv1.1 and Kv1.2 via a posttranscriptional mechanism, resulting in a reduction in the capacity of axonal D-type current to limit glutamate release, thus contributing to epileptogenesis.LGI1 | Kv1 channels | D-type current | intrinsic excitability | epilepsy

Synaptic core complex formation is an essential step in exocytosis, and assembly into a superhelical structure may drive synaptic vesicle fusion. To ascertain how Ca 2؉ could regulate this process, we examined calmodulin binding to recombinant core complex components. Surface plasmon resonance and pull-down assays revealed Ca 2؉ -dependent calmodulin binding (Kd ‫؍‬ 500 nM) to glutathione Stransferase fusion proteins containing synaptobrevin (VAMP 2) domains but not to syntaxin 1 or synaptosomal-associated protein of 25 kDa (SNAP-25). Deletion mutations, tetanus toxin cleavage, and peptide synthesis localized the calmodulin-binding domain to VAMP 77-94, immediately C-terminal to the tetanus toxin cleavage site (Q76-F77). In isolated synaptic vesicles, Ca 2؉ ͞calmodulin protected native membrane-inserted VAMP from proteolysis by tetanus toxin. Assembly of a 35 S-SNAP-25, syntaxin 1 GST-VAMP1-96 complex was inhibited by Ca 2؉ ͞calmodulin, but assembly did not mask subsequent accessibility of the calmodulin-binding domain. The same domain contains a predicted phospholipid interaction site. SPR revealed calcium-independent interactions between VAMP77-94 and liposomes containing phosphatidylserine, which blocked calmodulin binding. Circular dichroism spectroscopy demonstrated that the calmodulin͞ phospholipid-binding peptide displayed a significant increase in ␣-helical content in a hydrophobic environment. These data provide insight into the mechanisms by which Ca 2؉ may regulate synaptic core complex assembly and protein interactions with membrane bilayers during exocytosis.T ransmitter release at the nerve terminal occurs via calciumdependent exocytosis of the contents of synaptic vesicles at the presynaptic plasma membrane. Synaptic vesicle fusion involves the assembly of a heterotrimeric synaptic core [soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE)] complex composed of the vesicle-associated membrane protein (VAMP 2 or synaptobrevin) and two predominantly plasma membrane proteins, syntaxin 1 and synaptosomal-associated protein of 25 kDa (SNAP-25) (1, 2). The fundamental importance of these components is underlined by the fact that the metalloprotease activities of the botulinum (BoNT) and tetanus (TeTx) neurotoxins cleave the synaptic SNARE proteins at well-defined sites and potently inhibit transmitter release (3,4). Structural studies have demonstrated that the synaptic SNARE complex forms a four-helical parallel bundle with a superhelical twist (5, 6). It has been proposed that the assembly of this structure in a trans configuration, i.e., at the interface between a docked synaptic vesicle and the plasma membrane, pulls the opposing membranes together and may ultimately drive bilayer fusion (7). In support of this hypothesis, reconstitution of purified v-SNARE and t-SNARE proteins into distinct vesicle populations led to an increase in lipid mixing between the two vesicle pools, indicative of fusion (8). Although assembly is thought to be initiated in a trans configurati...

Three isoforms of synaptotagmin, a synaptic vesicle protein involved in neurotransmitter release, have been characterized in the rat, although functional differences between these isoforms have not been reported. In situ hybridization was used to define the localization of synaptotagmin I, II, and III transcripts in the rat CNS and pituitary and adrenal glands. Each of the three synaptotagmin genes has a unique expression pattern. The synaptotagmin III gene is expressed in most neurons, but transcripts are much less abundant than the products of the synaptotagmin I and II genes. A majority of neurons in the forebrain expressed both synaptotagmin I and III mRNAs while synaptotagmin II gene expression was confined to subsets of neurons in layers IV-VI of the cerebral cortex, in the dentate granule cell region, the hilus, and the CA1-CA3 areas of the hippocampus. In the cerebellum, all three transcripts were visualized in the granule cell layer. Furthermore, synaptotagmin I probes revealed striking differences between distinct populations of neurons, as in addition to moderate labeling of granule cells, much more prominent hybridization signals were detected on scattered cell bodies likely to be Golgi interneurons. In the most caudal part of the brain, synaptotagmin II transcripts were abundant and were coexpressed with synaptotagmin III mRNAs. This pattern was found in putative motoneurons of the spinal cord, suggesting that the two isoforms might be involved in exocytosis at the neuromuscular junction. Only synaptotagmin I mRNAs were detected in the anterior and intermediate pituitary and in adrenal medullary cells. These data reveal an unexpectedly subtle segregation of the expression of synaptotagmin genes and the existence of multiple combinations of synaptotagmin isoforms which may provide diversity in the regulation of neurosecretion.