R-type voltage-gated Ca(2+) channel interacts with synaptic proteins and recruits synaptotagmin to the plasma membrane of Xenopus oocytes.
It is well established that syntaxin 1A, synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptotagmin either alone or in combination, modulate the kinetic properties of voltage-gated Ca(2+) channels Ca(v)1.2 (Lc-channel) Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type). The interaction interface was found to reside at the cytosolic II-III domain of the alpha1 subunit of the channels. In this study, we demonstrated a functional coupling of human neuronal Ca(v)2.3 (R-type channel) with syntaxin 1A, SNAP-25 and synaptotagmin in BAPTA injected Xenopus oocytes. The kinetic properties of Ca(v)2.3 assembled with syntaxin 1A, SNAP-25 or synaptotagmin individually differed from Ca(v)2.3 associated with binary complexes syntaxin 1A/SNAP-25, syntaxin 1A/synaptotagmin or SNAP-25/synaptotagmin. Co-expression of Ca(v)2.3 with syntaxin 1A, SNAP-25 and synaptotagmin together, produced a channel with distinctive kinetic properties analogous to excitosome multiprotein complex generated by Ca(v)1.2 and Ca(v)2.2. Exchanging the current-carrying ions altered the kinetics of channel/synaptic proteins interaction, indicating a tight crosstalk formed between the permeation pathway of Ca(v)2.3 and the fusion apparatus during membrane depolarization. This putative coupling could predict how the release site might be organized to allow a rapid communication between the channel and the release machinery. In vivo confocal imaging of oocytes revealed GFP-synaptotagmin at the plasma membrane when the channel was present, as opposed to random distribution in its absence, consistent with Ca(2+)-independent molecular link of synaptotagmin and the channel. Synaptotagmin was detected at the membrane also in oocytes co-expressing the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Both imaging studies and protein-protein interactions in Xenopus oocytes show that channel linkage to synaptotagmin precedes Ca(2+) influx. Altogether, the R-type channel appears to associate with synaptic proteins to generate a multiprotein excitosome complex prior to Ca(2+)-entry. We propose that the distinct kinetics of the Ca(2+)-channel acquired by the close association with the vesicle and the t-SNAREs within the excitosome complex may be essential for depolarization evoked transmitter release.
Department of Biological Chemistry, The Silverman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
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Atlas
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism* / Q000494:pharmacology; D001464:Barium / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D020908:Calcium Channels, R-Type / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism* / Q000494:pharmacology; D027682:Cation Transport Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004558:Electric Stimulation / Q000379:methods; D005455:Fluorescent Antibody Technique / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism* / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism* / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism* / Q000494:pharmacology; D009865:Oocytes / Q000166:cytology*; D018408:Patch-Clamp Techniques / Q000379:methods; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D050827:Syntaxin 1; D014981:Xenopus
D000954:Antigens, Surface; C423009:CACNA1E protein, human; D015220:Calcium Channels; D020908:Calcium Channels, R-Type; D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; D007202:Indicators and Reagents; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C494208:SNAP25 protein, human; C494212:STX1A protein, human; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D050857:Synaptotagmins; D049452:Green Fluorescent Proteins; D001464:Barium; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium