Witcher D R, De Waard M, Campbell K P
Howard Hughes Medical Institute, Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242.
Neuropharmacology. 1993 Nov;32(11):1127-39. doi: 10.1016/0028-3908(93)90007-p.
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.
一种功能性N型钙离子通道（ω-芋螺毒素GVIA受体）已从兔脑中纯化出来，结果显示它由分子量分别为230K（α1B）、160K（α2δ）、95K和57K（β3）的四个亚基组成[Witcher D. R., De Waard M., Sakamoto J., Franzini-Armstrong C., Pragnell M., Kahl S.D.和Campbell K. D.（1993年）《科学》261: 486 - 489]。这四个亚基在蔗糖密度梯度上以单一复合物形式迁移，并通过亚基特异性多克隆抗体进行鉴定。针对纯化的受体复合物的多克隆抗体可免疫沉淀溶解的粗制兔脑膜中超过90%的[125I]ω-芋螺毒素GVIA（ω-CgTx）结合位点。此外，针对复合物中两个克隆亚基（α1B和β3）的独特GST融合蛋白亲和纯化的多克隆抗体可特异性免疫沉淀[125I]ω-CgTx结合位点，而不是[3H]PN200 - 110结合位点。对纯化的N型钙离子通道进行[125I]ω-CgTx结合分析表明，平衡结合对阳离子浓度升高敏感。钙和钡的IC50分别为2.5和5 mM。加入50 mM钡后15分钟内，[125I]ω-CgTx结合未显著降低。然而，对用10 μM ω-CgTx预孵育的纯化N型钙离子通道进行单通道分析表明，在存在50 mM钡和0.5 μM ω-CgTx的情况下，可检测到通道活性，但开放状态概率较低（P < 0.10）。这些数据表明，钙离子结合位点对ω-CgTx结合位点具有变构调节作用。由于在存在ω-CgTx的情况下通道门控持续存在，ω-CgTx结合位点可能不在通道孔内，并且可能与孔内钙离子结合位点不同。
