N-type calcium channel/syntaxin/SNAP-25 complex probed by antibodies to II-III intracellular loop of the alpha1B subunit.
Neuronal voltage-dependent calcium channels are integral components of cellular excitation and neurosecretion. In addition to mediating the entry of calcium across the plasma membrane, both N-type and P/Q-type voltage-dependent 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 structural analyses of N-type calcium channels have identified residues amino acids 718-963 as the site in the rat alpha1B subunit that mediates binding to syntaxin, synaptosome-associated protein of 25,000 mol. wt and synaptotagmin [Sheng et al. (1996) Nature 379, 451-454]. The purpose of this study was to employ site-directed antibodies to target domains within and outside of the interaction site on the rat alpha1B to probe potential binding sites for syntaxin/SNAP-25/synaptotagmin. Our results demonstrate that both antibodies employed in this study have access to their epitopes on the alpha1B as evidenced by equivalent immunoprecipitation of native [125I]omega-conotoxin GVIA-labeled alpha1B protein from CHAPS-solubilized preparations. The N-type voltage-dependent calcium channel immunoprecipitated by Ab CW14, the antibody directed to a domain outside of the synprint site, is associated with syntaxin and SNAP-25 with the recovery of these proteins, increasing in parallel to the recovery of alpha1B. However, when we used the antibody raised to an epitope within the synprint site (Ab CW8) to immunoprecipitate N-type calcium channels, the alpha1B was depleted of more than 65% of syntaxin and 80% of SNAP-25 when compared to the recovery of these proteins using Ab CW14. This is the first report of a defined epitope on the alpha1B subunit II-III loop (amino acids 863-875) whose perturbation by a site-directed antibody influences the dissociation of SNAP-25 and syntaxin.
Vance
C L
CL
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4970, USA.
Begg
C M
CM
Lee
W L
WL
Dubel
S J
SJ
Copeland
T D
TD
Sönnichsen
F D
FD
McEnery
M W
MW
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D017433:Protein Structure, Secondary; D050765:Qa-SNARE Proteins; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins
D000906:Antibodies; D015220:Calcium Channels; D002135:Calcium-Binding Proteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D050765:Qa-SNARE Proteins; C494208:SNAP25 protein, human; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D002118:Calcium