title: Mechanism of SNARE protein binding and regulation of Cav2 channels by phosphorylation of the synaptic protein interaction site.
abstract: Ca(v)2.1 and Ca(v)2.2 channels conduct P/Q-type and N-type Ca(2+) currents that initiate neurotransmission and bind SNARE proteins through a synaptic protein interaction (synprint) site. PKC and CaMKII phosphorylate the synprint site and inhibit SNARE protein binding in vitro. Here we identify two separate microdomains that each bind syntaxin 1A and SNAP-25 in vitro and are regulated by PKC phosphorylation at serines 774 and 898 and CaMKII phosphorylation at serines 784 and 896. Activation of PKC resulted in its recruitment to and phosphorylation of Ca(V)2.2 channels, but PKC phosphorylation did not dissociate Ca(V)2.2 channel/syntaxin 1A complexes. Chimeric Ca(V)2.1a channels containing the synprint site of Ca(v)2.2 gain modulation by syntaxin 1A, which is blocked by PKC phosphorylation at the sites identified above. Our results support a bipartite model for the synprint site in which each SNARE-binding microdomain is controlled by a separate PKC and CaMKII phosphorylation site that regulates channel modulation by SNARE proteins.
authors.affiliation: Department of Pharmacology, University of Washington, Seattle, WA 98195, USA.
authors.forename: Charles T
authors.initials: CT
authors.lastname: Yokoyama
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Myers
authors.forename: Jian
authors.initials: J
authors.lastname: Fu
authors.forename: Susan M
authors.initials: SM
authors.lastname: Mockus
authors.forename: Todd
authors.initials: T
authors.lastname: Scheuer
authors.forename: William A
authors.initials: WA
authors.lastname: Catterall
mesh_terms: D000954:Antigens, Surface / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D002490:Central Nervous System / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D050600:SNARE Proteins; D013569:Synapses / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: D000954:Antigens, Surface; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011493:Protein Kinase C; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; C500545:CACNA1B protein, human; C494208:SNAP25 protein, human; C494212:STX1A protein, human