[Dosage and specificity of anti-calcium channel antibodies in Lambert-Eaton myasthenic syndrome]. / Dosage et spécificité d'autoanticorps anti-canaux calcium dans le syndrome myasthénique de Lambert-Eaton.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune channelopathy in which patients produce autoantibodies directed against voltage-gated calcium channels. Autoantibodies down-regulate calcium channels resulting in reduced transmitter release, which in turn leads to muscular weakness and autonomic dysfunction. LEMS is paraneoplastic in 60-70% of patients, most frequently associated with small cell lung carcinoma (SCLC). SCLC lines express many neuronal and neuroendocrine proteins including neuronal calcium channels of the Cav2 family (P/Q and N-type channels). It is thus likely that the paraneoplastic form of LEMS is the consequence of an anti-tumoral immune response and the production of antibodies that cross-react with identical or homologous antigens in nerve terminals. Neurological symptoms generally appear several Months before detection of the tumor. Consequently correct diagnosis of LEMS is crucial as it can allow early treatment of a particularly aggressive carcinoma. Based on published studies, our laboratory has set-up serological assays for LEMS autoantibodies as an aid to diagnosis. Calcium channels in detergent extracts of rat brain or cerebellum membranes were labeled with radioligands specific for N-type (125I-omega conotoxin GVIA) or P/Q-type (125I-omega conotoxin MVIIC) calcium channels. Autoantibodies that immunoprecipitate the ligand/channel complex can thus be titrated. Analysis of 31 LEMS sera revealed the presence of anti-N type channel antibodies in 58% and anti-P/Q type channel antibodies in 74% of patients with titres ranging from 90 to 2950 pM. Only 5 patients were seronegative in both tests, thus a combination of the two assays reliably detected autoantibodies in 26/31 (84%) patients.

alpha-Latrotoxin, acting via two Ca2+-dependent pathways, triggers exocytosis of two pools of synaptic vesicles.

alpha-Latrotoxin stimulates three types of [(3)H]gamma-aminobutyric acid and [(14)C]glutamate release from synaptosomes. The Ca(2+)-independent component (i) is insensitive to SNAP-25 cleavage or depletion of vesicle contents by bafilomycin A1 and represents transmitter efflux mediated by alpha-latrotoxin pores. Two other components of release are Ca(2+)-dependent and vesicular but rely on distinct mechanisms. The fast receptor-mediated pathway (ii) involves intracellular Ca(2+) stores and acts upon sucrose-sensitive readily releasable vesicles; this mechanism is insensitive to inhibition of phosphatidylinositol 4-kinase (PI 4-kinase). The delayed pore-dependent exocytotic component (iii) is stimulated by Ca(2+) entering through alpha-latrotoxin pores; it requires PI 4-kinase and occurs mainly from depot vesicles. Lanthanum perturbs alpha-latrotoxin pores and blocks the two pore-mediated components (i, iii) but not the receptor-mediated release (ii). alpha-Latrotoxin mutant (LTX(N4C)) cannot form pores and stimulates only the Ca(2+)-dependent receptor-mediated amino acid exocytosis (ii) (detectable biochemically and electrophysiologically). These findings explain experimental data obtained by different laboratories and implicate the toxin receptors in the regulation of the readily releasable pool of synaptic vesicles. Our results also suggest that, similar to noradrenergic vesicles, amino acid-containing vesicles at some point in their cycle require PI 4-kinase.

Role of Thr(11) in the binding of omega-conotoxin MVIIC to N-type Ca2+ channels.

As replacement of Thr(11) of omega-conotoxin MVIIC with Ala significantly reduced the affinity for both N- and P/Q-type calcium channels, we examined the effect of substitution at this position with other residues. Binding assays using rat cerebellar P2 membranes showed that the affinity is in the order of Leu>Val, aminobutyric acid, Thr>Asn&z.Gt;Ser, Ala, Asp, Phe, Tyr for N-type channels and Thr>Leu, Val, aminobutyric acid, Asn, Ser>Ala&z.Gt;Asp, Phe, Tyr for P/Q-type channels, suggesting that aliphatic amino acids with longer side chains are favorable for block of N-type channels. The effects of substitution were examined electrophysiologically in BHK cells expressing N-type Ca2+ channels. Inhibition of Ba2+ current by the analogs did not completely correlate with binding affinity, although binding to BHK cells was comparable to rat cerebellar membranes.

[Calcium channels and migraine]. / Canaux calciques et migraine.

Familial hemiplegic migraine is a hereditary form of migraine in which the aura includes a certain degree of motor deficit. A first gene responsible for this disease was located on chromosome 19 in 1993, and identified in 1996. It encodes the principal alpha 1-subunit of a potential-dependent calcium channel, the P/Q channel, which is selectively expressed in the nervous system. This channel is particularly rich in nerve terminals, where it contributes to the triggering and release of neuromediators. In patients with hemiplegic migraine, mis-sense mutations have been detected which result in a modification of channel function. Other mutations which lead to the synthesis of inactive protein truncations have been described in another disease, type 2 episodic ataxia. In the mouse, mutations in the same gene lead to different phenotypes (tottering mouse, leaner mouse). Finally, possible links between P/Q calcium channel dysfunction and migraine have been discussed.

alpha-latrotoxin forms calcium-permeable membrane pores via interactions with latrophilin or neurexin.

In order to explore the mechanisms by which alpha-latrotoxin activates neurotransmitter release, we have characterized its effects by patch-clamp methods on cells heterologously expressing its receptors, latrophilin-1 or neurexin-Ialpha. Application of alpha-latrotoxin (1 nM) to cells expressing rat latrophilin or neurexin, but not mock-transfected cells, induced a cationic conductance. In cells expressing latrophilin, current development was slow in the absence of divalent cations, but was accelerated by Ca2+ or Mg2+. In cells expressing neurexin, alpha-latrotoxin did not elicit currents in the absence of Ca2+. The toxin-induced conductance was rectifying, persistent, permeable to monovalent and divalent cations, but blocked by La3+. Single-channel recording revealed a permanently open state, with the same unitary conductance irrespective of whether cells expressed latrophilin or neurexin. Therefore, while pore formation displayed differences consistent with the reported properties of alpha-latrotoxin binding to latrophilin and neurexin, the pores induced by alpha-latrotoxin had identical properties. These results suggest that after anchoring to either of its nerve terminal receptors, alpha-latrotoxin inserts into the membrane and constitutes a single type of transmembrane ion pore.

Humoral immunity against glutamic acid decarboxylase and tyrosine phosphatase IA-2 in Lambert-Eaton myasthenic syndrome.

Some beta-cell-specific autoantigens also are present in the central nervous system. Furthermore, stiff man syndrome, an autoimmune neurological disease, is frequently associated with diabetes and shares with this one an anti-GAD and IA-2 humoral immunoreactivity. We wondered whether these autoantibodies could be found in other neurological diseases with a present or supposed autoimmune origin. So, anti-GAD65 (GAD65A) and anti-IA-2 (IA-2A) autoantibodies were assayed in various neurological diseases. There was a higher prevalence of such antibodies in Lambert-Eaton myasthenic syndrome (LEMS) (GAD65A, 35%; IA-2A, 21%; double positivity, 18%) compared to amyotrophic lateral sclerosis (18%, 12%, and 12%, respectively) and multiple sclerosis (10%, 3%, and 3%, respectively). In LEMS, the humoral reaction was more frequent and/or appeared earlier in the paraneoplastic forms. The detection of such autoantibodies in patients with small-cell lung carcinoma (SCLC) without LEMS suggests that these autoantigens, GAD65 and IA-2, could be produced by SCLC tissue.

Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin.

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 (K(d) = 500 nM) to glutathione S-transferase 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 (Q(76)-F(77)). 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-VAMP(1-96) 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 VAMP(77-94) and liposomes containing phosphatidylserine, which blocked calmodulin binding. Circular dichroism spectroscopy demonstrated that the calmodulin/phospholipid-binding peptide displayed a significant increase in alphahelical 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.

Synaptotagmins in membrane traffic: which vesicles do the tagmins tag?

The aim of this review is to give a broad picture of what is actually known about the synaptotagmin family. Synaptotagmin I is an abundant synaptic vesicle and secretory granule protein in neurons and endocrine cells which plays a key role in Ca(2+)-induced exocytosis. It belongs to the large family of C2 domain-proteins as it contains two internal repeats that have homology to the C2 domain of protein kinase C. Eleven synaptotagmin genes have been described in rat and mouse. Except for synaptotagmin I, and by analogy synaptotagmin II, the functions of these proteins are unknown. In this review we focus on data obtained on the various isoforms without exhaustively discussing the role of synaptotagmin I in neurotransmission. Numerous in vitro interactions of synaptotagmin I with key components of the exocytosis-endocytosis machinery have been reported. Additional data concerning the other synaptotagmins are now becoming available and are reviewed here. Only interactions which have been described for several synaptotagmins, are mentioned. It is unlikely that a single isoform displays all of these potential interactions in vivo and probably the subcellular distribution of the protein will favor some of them and preclude others. Therefore, to discuss the putative role of the various synaptotagmins we have examined in detail published data concerning their localization.

Synaptotagmin I and IV define distinct populations of neuronal transport vesicles.

Mammalian synaptotagmins constitute a multigene family of at least 11 membrane proteins. We have characterized synaptotagmin IV using antibodies directed against the C2A domain of the protein. Antibodies reacted specifically with a protein band that migrated as a 41-44 kDa doublet. Synaptotagmin IV expression was regulated throughout development. A strong decrease in the amount detected by Western blotting occurred between postnatal day 5 and adulthood, in agreement with studies on the expression of synaptotagmin IV transcripts. In subcellular fractionation, synaptotagmin IV was not detected in the synaptic vesicle-enriched fraction. Immunofluorescence microscopy was concordant with this finding. In 6-day-old rat cerebellum and cultured hippocampal neurons the subcellular distribution of synaptotagmin IV was clearly different from that of synaptotagmin I. Synaptotagmin IV displayed a punctate non-polarized distribution on neuronal extensions, whereas synaptotagmin I staining was essentially synaptic. Synaptotagmin IV staining was also observed in the soma in strong perinuclear fluorescent puncta superimposed on that of Golgi/TGN markers. Furthermore, synaptotagmin IV was seen in the proximal part of the growth cone domain and not in the microfilament-rich region which includes filopodia. Co-localizations with the adhesion molecules vinculin and zyxin at the proximal part of growth cones were observed. Synaptotagmin IV may thus be involved in the regulation of specific membrane-trafficking pathways during brain development.

Binding of six chimeric analogs of omega-conotoxin MVIIA and MVIIC to N- and P/Q-type calcium channels.

Replacement of the N-terminal half of omega-conotoxin MVIIC, a peptide blocker of P/Q-type calcium channels, with that of omega-conotoxin MVIIA significantly increased the affinity for N-type calcium channels. To identify the residues essential for subtype selectivity, we examined single reverse mutations from MVIIA-type to MVIIC-type in this chimeric analog. A reverse mutation from Lys(7) to Pro(7) decreased the affinity for both P/Q- and N-type channels, whereas that from Leu(11) to Thr(11) increased the affinity for P/Q-type channels and decreased the affinity for N-type channels. The roles of these two residues were confirmed by synthesizing two MVIIC analogs in which Pro(7) and Thr(11) were replaced with Lys(7) and Leu(11), respectively.

Direct interaction between synaptotagmin and the intracellular loop I-II of neuronal voltage-sensitive sodium channels.

Synaptotagmin, a synaptic vesicle protein involved in Ca(2+)-regulated exocytosis, displayed direct high affinity interaction with neuronal sodium channels. Monoclonal antibodies directed against synaptotagmins I and II adsorbed in a concentration-dependent and -specific manner [(3)H]saxitoxin prelabeled sodium channels extracted with detergent from nerve endings. Conversely, co-immunoprecipitation of synaptotagmin was achieved by antibodies against sodium channel subunits. Consistent with the co-immunoprecipitation assays, solubilized [(3)H]saxitoxin-prelabeled sodium channels were trapped on immobilized maltose binding protein (MBP)-synaptotagmin I. In vitro recombinant protein assays were employed to identify the interaction site of synaptotagmin I, which was located on the cytoplasmic loop between domains I and II of the sodium channel alphaIIA subunit. The co-immunoprecipitated synaptotagmin-sodium channel complexes were found to be Ca(2+)-dependent; this effect was mimicked by Ba(2+) and Sr(2+) but not Mg(2+). Finally the complex was shown to be distinct from the synaptotagmin-SNARE protein complex that can selectively interact with presynaptic calcium channels (N and P/Q types). Thus, our findings demonstrate an unexpected and direct interaction between sodium channels and synaptotagmin. The Ca(2+)-regulated association between sodium channels and a protein implicated in vesicular fusion may have intriguing consequences for the establishment and regulation of neuronal excitability.

Binding of Ala-scanning analogs of omega-conotoxin MVIIC to N- and P/Q-type calcium channels.

omega-Conotoxin MVIIC binds to P/Q-type calcium channels with high affinity and N-type channels with low affinity. To reveal the residues essential for subtype selectivity, we synthesized Ala-scanning analogs of MVIIC. Binding assays using rat cerebellar P(2) membranes suggested that Thr(11), Tyr(13) and Lys(2) are essential for binding to both N- and P/Q-type channels, whereas Lys(4) and Arg(22) are important for binding to P/Q-type channels. These results suggest that MVIIC interacts with P/Q-type channels via a large surface, in good agreement with previous observations using chimeric analogs.

Calcium-dependent dissociation of synaptotagmin from synaptic SNARE complexes.

The formation of the synaptic core (SNARE) complex constitutes a crucial step in synaptic vesicle fusion at the nerve terminal. The interaction of synaptotagmin I with this complex potentially provides a means of conferring Ca2+-dependent regulation of exocytosis. However, the subcellular compartments in which interactions occur and their modulation by Ca2+ influx remain obscure. Sodium dodecyl sulfate (SDS)-resistant core complexes, associated with synaptotagmin I, were enriched in rat brain fractions containing plasma membranes and docked synaptic vesicles. Depolarization of synaptosomes triggered [3H]GABA release and Ca2+-dependent dissociation of synaptotagmin from the core complex. In perforated synaptosomes, synaptotagmin dissociation was induced by Ca2+ (30-300 microM) but not Sr2+ (1 mM); it apparently required intact membrane bilayers but did not result in disassembly of trimeric SNARE complexes. Synaptotagmin was not associated with unstable v-SNARE/t-SNARE complexes, present in fractions containing synaptic vesicles and cytoplasm. These complexes acquired SDS resistance when N-ethylmaleimide-sensitive fusion protein (NSF) was inhibited with N-ethylmaleimide or adenosine 5'-O-(3-thiotriphosphate), suggesting that constitutive SNARE complex disassembly occurs in undocked synaptic vesicles. Our findings are consistent with models in which the Ca2+ triggered release of synaptotagmin precedes vesicle fusion. NSF may then dissociate ternary core complexes captured by endocytosis and recycle/prime individual SNARE proteins.

Redistribution of presynaptic proteins during alpha-latrotoxin-induced release of neurotransmitter and membrane retrieval at the frog neuromuscular junction.

Calcium-dependent exocytosis at the nerve terminal involves the synaptic core (SNARE) complex composed of the t-SNAREs syntaxin 1 and synaptosome-associated protein of 25 kDa (SNAP-25), and the v-SNARE vesicle-associated membrane protein (VAMP/synaptobrevin), a stable heterotrimer which can associate with the putative calcium sensor protein, synaptotagmin. The distribution of these proteins at the frog neuromuscular junction was examined by immunofluorescent staining and confocal microscopy following exocytosis induced by alpha-latrotoxin. Experiments were performed under conditions in which synaptic vesicle recycling was either maintained in balance with exocytosis, or completely blocked, or during recovery from block of endocytosis. When endocytosis was maintained, protein distribution was essentially identical to that of unstimulated nerve terminals, in which syntaxin 1 and SNAP-25 are localized to the presynaptic active zones coincident with the postsynaptic folds that contain a high density of acetylcholine receptors (AChRs). Block of endocytosis led to complete incorporation of vesicle proteins into the plasmalemma, and t-SNARE distribution was no longer restricted to active zones. Five minutes after the onset of recovery, both synaptic vesicle proteins and t-SNARE proteins were concentrated into small spots, in a similar pattern to that obtained following endocytosis of the vital styryl dye FM1-43. These findings are consistent with a model in which following sustained exocytosis, t-SNARE trafficking involves internalization and transit via a vesicular compartment before recycling to the presynaptic plasma membrane.

Antibodies against the beta subunit of voltage-dependent calcium channels in Lambert-Eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome is an autoimmune disease that impairs neuromuscular transmission. Several studies suggest that neurotransmitter release is reduced by an immune response directed against the calcium channel complex of nerve terminals. The immunoglobulin G fractions from Lambert-Eaton myasthenic syndrome patients immunoprecipitate solubilized neuronal N- and P/Q-type channels and in certain cases brain, skeletal and cardiac muscle L-type channels [El Far O. et al. (1995) J. Neurochem. 64, 1696-1702; Lennon V. A. and Lambert E. H. (1989) Mayo Clin. Proc. 64, 1498-1504; Sher E. et al. (1989) Lancet ii, 640-643; Suenaga A. et al. (1996) Muscle Nerve 19, 1166-1168]. These channel immunoprecipitation assays are considered as useful for the diagnosis of this syndrome. In this study, we demonstrate that two predominant neuronal voltage-dependent calcium channel beta subunits (beta3 and beta4, of mol. wt 58,000) are general targets of Lambert-Eaton myasthenic syndrome autoantibodies. Of 20 disease sera tested, 55% were able to immunoprecipitate 35S-labeled beta subunits. All five patients affected with small-cell lung carcinoma were positive for the beta-subunit immunoprecipitation assay. Interestingly, only a fraction of the beta-subunit-positive sera was also able to immunoprecipitate N- and P/Q-type channels, suggesting that several of the beta-subunit epitopes are masked in native channels. In accordance with this observation, we found that several beta-positive sera were able to prevent the interaction between calcium channel alpha1 and beta subunits in vitro. In cases where sera were able to immunoprecipitate beta subunits, N- and P/Q-type channels, the immunoprecipitation of both channel types was either partially or entirely mediated by beta-subunit antibodies. Our results suggest that assays based on the immunoprecipitation of beta subunits can be used as an additional test to assist in the diagnosis of Lambert-Eaton myasthenic syndrome.

Interactions between proteins implicated in exocytosis and voltage-gated calcium channels.

Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium influx through P/Q-type or N-type calcium channels. Purification of N-type channels from rat brain synaptosomes initially suggested molecular interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experiments were consistent with the hypothesis that both N- and P/Q-type calcium channels, but not L-type channels, are associated with the 7S complex containing syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immunofluorescence confocal microscopy at the frog neuromuscular junction confirmed that calcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experiments with recombinant proteins were performed to map synaptic protein interaction sites on the alpha 1A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the alpha 1A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium influx close to the channel mouth. Cysteine string proteins (CSPs) contain a J-domain characteristic of molecular chaperones that cooperate with Hsp70. They are located on synaptic vesicles and thought to be involved in modulating the activity of presynaptic calcium channels. CSPs were found to bind to the same domain of the calcium channel as synaptotagmin, and also to associate with VAMP. CSPs may act as molecular chaperones in association with Hsp70 to direct assembly or dissociation of multiprotein complexes at the calcium channel.

Interactions between presynaptic calcium channels and proteins implicated in synaptic vesicle trafficking and exocytosis.

Monoclonal antibodies were generated by immunizing mice with chick brain synaptic membranes and screening for immunoprecipitation of solubilized omega conotoxin GVIA receptors (N-type calcium channels). Antibodies against two synaptic proteins (p35--syntaxin 1 and p58--synaptotagmin) were produced and used to purify and characterize a ternary complex containing N-type channels associated with these two proteins. These results provided the first evidence for a specific interaction between presynaptic calcium channels and SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. Immunoprecipitation experiments supported the conclusion that syntaxin 1/SNAP-25/VAMP/synaptotagmin I or II complexes associate with N-type, P/Q-type, but not L-type calcium channels from rat brain nerve terminals. Immunofluorescent confocal microscopy at the frog neuromuscular junction was consistent with the co-localization of syntaxin 1, SNAP-25, and calcium channels, all of which are predominantly expressed at active zones of the presynaptic plasma membrane facing post-synaptic folds rich in acetylcholine receptors. The interaction of proteins implicated in calcium-dependent exocytosis with presynaptic calcium channels may locate the sensor(s) that trigger vesicle fusion within a microdomain of calcium entry.

Interaction of cysteine string proteins with the alpha1A subunit of the P/Q-type calcium channel.

Cysteine string proteins (Csps) are J-domain chaperone proteins anchored at the surface of synaptic vesicles. Csps are involved in neurotransmitter release and may modulate presynaptic calcium channel activity, although the molecular mechanisms are unknown. Interactions between Csps, proteins of the synaptic core (SNARE) complex, and P/Q-type calcium channels were therefore explored. Co-immunoprecipitation suggested that Csps occur in complexes containing synaptobrevin (VAMP), but not syntaxin 1, SNAP-25, nor P/Q-type calcium channels labeled with 125I-omega-conotoxin MVIIC. However binding experiments with 35S-labeled Csp1 demonstrated an interaction (apparent KD = 700 nM at pH 7.4 and 4 degreesC) with a fusion protein containing a segment of the cytoplasmic loop linking homologous domains II-III of the alpha1A calcium channel subunit (BI isoform, residues 780-969). Binding was specific as it was displaced by unlabeled Csp1, and no interactions were detected with fusion proteins containing other calcium channel domains, VAMP, or syntaxin 1A. A Csp binding site on the P/Q-type calcium channel is thus located within the 200 residue synaptic protein interaction site that can also bind syntaxin I, SNAP-25, and synaptotagmin I. Csp may act as a molecular chaperone to direct assembly or disassembly of exocytotic complexes at the calcium channel.

Binding of chimeric analogs of omega-conotoxin MVIIA and MVIIC to the N- and P/Q-type calcium channels.

Despite their high sequence homology, the peptide neurotoxins omega-conotoxin MVIIA and MVIIC selectively block N- and P/Q-type calcium channels, respectively. To study the recognition mechanism of calcium channel subtypes, two chimeric analogs of omega-conotoxin MVIIA and MVIIC were synthesized by exchanging their N- and C-terminal halves. Binding assay for both N- and P/Q-type calcium channels showed that amino acid residues restricted to the N-terminal half are important for the recognition of N-type channels, whereas essential residues for P/Q-type channel recognition are widely spread over the whole omega-conotoxin molecule.

Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the alpha1A subunit of the P/Q-type calcium channel.

Synaptotagmins are synaptic vesicle proteins containing two calcium-binding C2 domains which are involved in coupling calcium influx through voltage-gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q-type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co-immunoprecipitated [125I]omega-conotoxin MVIIC-labelled calcium channels. Direct interactions were studied between in vitro-translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the alpha1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780-969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium-independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4 degrees C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore-forming subunit of the P/Q-type channel may optimally locate the calcium-binding sites that initiate exocytosis within a zone of voltage-gated calcium entry.