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

纯化的 N 型 Ca2+ 通道的表征以及 ω-芋螺毒素 GVIA 结合的阳离子敏感性

DOI：

10.1016/0028-3908(93)90007-p

发表时间：

1993

期刊：

Neuropharmacology

影响因子：

4.7

通讯作者：

中科院分区：

医学2区

文献类型：

作者：

D. Witcher;M. Waard;K. Campbell

张建军，张建军，张建军，等。从兔脑中分离得到的n型钙离子通道（ω-conotoxin GVIA receptor）由分子量为230 K (α1B), 160 K (α2δ)， 95 K和57 K （β3）的4个亚基组成[J].科学通报，1993,26(1):486-489。这四个亚基作为一个单一的复合物在蔗糖密度梯度上迁移，并被亚基特异性多克隆抗体识别。针对纯化受体复合物的多克隆抗体可在溶解的粗兔脑膜中沉淀90%以上的[125I]ω- concontoxin GVIA （ω-CgTx）结合位点。此外，从两个克隆的复合物亚基（α1Bandβ3）中获得的针对GST融合蛋白的亲和纯化多克隆抗体特异性地免疫沉淀了[125I]ω-CgTx结合位点，而不是[3H]PN200-110结合位点。[125I]ω-CgTx与纯化的n型Ca2+通道的结合分析表明，平衡结合对阳离子浓度的增加很敏感。钙和钡的ic50分别为2.5 mM和5 mM。在加入50 mM钡后的15 min内，[125II]ω-CgTx结合没有明显降低。然而，对10 μM ω-CgTx预孵育的纯化n型Ca2+通道的单通道分析表明，在50 mM钡和0.5 μM ωCgTx的存在下，通道活性被检测到，但打开状态概率很低（P< 0.10）。这些数据表明Ca2+结合位点(s)变构调节ω-CgTx结合位点。由于通道门控在ω-CgTx存在的情况下持续存在，ω-CgTx结合位点可能不在通道的孔内，并且可能与孔内Ca2+结合位点不同。

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)Science261: 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 (α1Bandβ3) 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 IC50for 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.