Characterization of the purified N-type Ca2+ channel and the cation sensitivity of ω-conotoxin GVIA binding

Abstract

A functional N-type Ca2+ channel (ω-conotoxin GVIA receptor) has been purified from rabbit brain and shown to be composed of four subunits of molecular weights 230 K (α1B), 160 K (α2δ), 95 K and 57 K (β3) [Witcher D. R., De Waard M., Sakamoto J., Franzini-Armstrong C., Pragnell M., Kahl S. D. and Campbell K. D. (1993) Science 261: 486-489]. These four subunits migrate on sucrose density gradients as a single complex and are identified by subunit specific polyclonal antibodies. Polyclonal antibodies against the purified receptor complex immunoprecipitate greater than 90% of the [125I]ω-conotoxin GVIA (ω-CgTx) binding sites in solubilized crude rabbit brain membranes. Furthermore, polyclonal antibodies affinity-purified against unique GST fusion proteins from two of the cloned subunits in the complex (α1B and β3) specifically immunoprecipitated [125I]ω-CgTx binding sites and not [3H]PN200-110 binding sites. Analysis of [125I]ω-CgTx binding to the purified N-type Ca2+ channel demonstrated that the equilibrium binding was sensitive to increasing cation concentrations. The IC50 for calcium and barium was 2.5 and 5 mM, respectively. [125II]ω-CgTx binding was not significantly reduced within 15 min after the addition of 50 mM barium. However, single channel analysis of the purified N-type Ca2+ channel preincubated with 10 μM ω-CgTx demonstrated that in the presence of 50 mM barium and 0.5 μM ωCgTx, channel activity was detected but at a low open state probability (P < 0.10). These data suggest that the Ca2+ binding site(s) allosterically regulates the ω-CgTx binding site. Since the channel gating persisted in the presence of ω-CgTx, the ω-CgTx binding site may not be located within the pore of the channel and may be different from intra-pore Ca2+ binding sites. © 1993.