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.
Cohen
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Department of Biological Chemistry, The Silverman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
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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