N-type calcium channel/syntaxin/SNAP-25 complex probed by antibodies to II-III intracellular loop of the alpha(1B) subunit

Neuronal voltage-dependent calcium channels are integral components of cell ular excitation and neurosecretion. In addition to mediating the entry of c alcium across the plasma membrane, both N-type and P/Q-type voltage-depende nt calcium channels have been shown to form stable complexes with synaptic vesicle and presynaptic membrane proteins, indicating a structural role for the voltage-dependent calcium channels in secretion. Recently, detailed st ructural analyses of N-type calcium channels have identified residues amino acids 718-963 as the site in the rat alpha(1B) subunit that mediates bindi ng to syntaxin, synaptosome-associated protein of 25,000 mol. wt and synapt otagmin [Sheng et al. (1996) Nature 379, 451-454]. The purpose of this stud y was to employ site-directed antibodies to target domains within and outsi de of the interaction site on the rat alpha(1B) to probe potential binding sites for syntaxin/SNAP-25/synaptotagmin. Our results demonstrate that both antibodies employed in this study have ac cess to their epitopes on the alpha(1B) as evidenced by equivalent immunopr ecipitation of native [I-125]omega-conotoxin GVIA-labeled alpha(1B) protein from CHAPS-solubilized preparations. The N-type voltage-dependent calcium channel immunoprecipitated by Ab CW14, the antibody directed to a domain ou tside of the synprint site, is associated with syntaxin and SNAP-25 with th e recovery of these proteins, increasing in parallel to the recovery of alp ha(1B). However, when we used the antibody raised to an epitope within the synprint site (Ab CW8) to immunoprecipitate N-type calcium channels, the al pha(1B) was depleted of more than 65% of syntaxin and 80% of SNAP-25 when c ompared to the recovery of these proteins using Ab CW14. This is the first report of a defined epitope on the alpha(1B) subunit II-III loop (amino aci ds 863-875) whose perturbation by a site-directed antibody influences the d issociation of SNAP-25 and syntaxin. (C) 1999 IBRO. Published by Elsevier S cience Ltd.