Characterization of the purified N-type Ca2+ channel and the cation sensitivity of omega-conotoxin GVIA binding.
A functional N-type Ca2+ channel (omega-conotoxin GVIA receptor) has been purified from rabbit brain and shown to be composed of four subunits of molecular weights 230 K (alpha 1B), 160 K (alpha 2 delta), 95 K and 57 K (beta 3) [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]omega-conotoxin GVIA (omega-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 (alpha 1B and beta 3) specifically immunoprecipitated [125I]omega-CgTx binding sites and not [3H]PN200-110 binding sites. Analysis of [125I]omega-CgTx binding to the purified N-type Ca2+ channel demonstrated that the equilibrium binding was sensitive to increasing cation concentration. The IC50 for calcium and barium was 2.5 and 5 mM, respectively. [125I]omega-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 microM omega-CgTx demonstrated that in the presence of 50 mM barium and 0.5 microM omega-CgTx, 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 omega-CgTx binding site. Since the channel gating persisted in the presence of omega-CgTx, the omega-CgTx binding site may not be located within the pore of the channel and may be different from intra-pore Ca2+ binding sites.
Witcher
D R
DR
Howard Hughes Medical Institute, Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242.
De Waard
M
M
Campbell
K P
KP
D000818:Animals; D001923:Brain Chemistry / Q000187:drug effects; D002121:Calcium Channel Blockers / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D004594:Electrophysiology; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D010455:Peptides / Q000378:metabolism* / Q000494:pharmacology; D011233:Precipitin Tests; D011817:Rabbits; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002413:Cations, Divalent; D010455:Peptides; D020866:omega-Conotoxin GVIA; D005982:Glutathione Transferase