title: The C2A domain of synaptotagmin alters the kinetics of voltage-gated Ca2+ channels Ca(v)1.2 (Lc-type) and Ca(v)2.3 (R-type).
abstract: Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.
authors.affiliation: Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Roy
authors.initials: R
authors.lastname: Cohen
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Elferink
authors.forename: Daphne
authors.initials: D
authors.lastname: Atlas
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry*; D020746:Calcium Channels, L-Type / Q000737:chemistry*; D020908:Calcium Channels, R-Type; D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; D003600:Cytosol / Q000378:metabolism; D004594:Electrophysiology; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008954:Models, Biological; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D011107:Polylysine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D050857:Synaptotagmins; D050827:Syntaxin 1; D013997:Time Factors; D014981:Xenopus
chemical_list: D000954:Antigens, Surface; C498917:Cacna1e protein, rat; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D020908:Calcium Channels, R-Type; D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; D020662:Guanine Nucleotide Exchange Factors; C419385:L-type calcium channel alpha(1C); D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; C494212:STX1A protein, human; C121631:SYT1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D050857:Synaptotagmins; D011107:Polylysine; D005982:Glutathione Transferase; D002118:Calcium