A recombinant inwardly rectifying potassium channel coupled to GTP-binding proteins.
A recombinant inwardly rectifying potassium channel coupled to GTP-binding proteins.
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DOI：
10.1085/jgp.107.3.381
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发表时间：
1996-03
影响因子：
3.8
通讯作者：
Logothetis, D E
中科院分区：
文献类型：
--
作者：
Chan, K W;Langan, M N;Sui, J L;Kozak, J A;Pabon, A;Ladias, J A;Logothetis, D E
GTP 结合 (G) 蛋白已被证明可介导心脏、神经元和神经内分泌细胞中内向整流钾 (K+) 通道的激活。在这里，我们报告了重组内向整流通道的功能表达，我们将其称为 KGP（或 hpKir3.4），表明它具有 K+ 选择性、G 蛋白门控并从人胰腺中分离出来。非洲爪蟾卵母细胞中的 KGP 表达导致相当大的基础（或激动剂非依赖性）电流，而与 G 蛋白连接受体共表达则产生额外的激动剂诱导电流。 KGP 和 hGIRK1（GIRK1/Kir3.1 的人脑同源物）的共表达产生的基础电流比单独使用 KGP 或 hGIRK1 观察到的基础电流大得多，并且在与受体共表达时，可以获得类似的大激动剂诱导电流。由于 KGP 或 KGP/hGIRK1 表达，百日咳毒素治疗显着减少了激动剂依赖性电流。有趣的是，PTX 还显着降低了基础 KGP 或 KGP/hGIRK1 电流，表明基础活性很大程度上也是 G 蛋白门控的结果。当这两个通道与受体共表达时，无论KGP和hGIRK1单独表达还是一起表达，激动剂引起的电流相对增加相似。当体外翻译或在非洲爪蟾卵母细胞或 CHO 哺乳动物细胞中表达时，KGP 产生非糖基化的 45-kD 蛋白质。针对 KGP 或 hGIRK1 的抗体使卵母细胞中共表达的两种蛋白共沉淀，为两个通道的异聚组装提供了证据，并表明当前两个通道亚基共表达所见的增强作用是由于它们之间的特异性相互作用。大小与 KGP 相似的内源卵母细胞蛋白也与 hGIRK1 共沉淀。
GTP-binding (G) proteins have been shown to mediate activation of inwardly rectifying potassium (K+) channels in cardiac, neuronal and neuroendocrine cells. Here, we report functional expression of a recombinant inwardly rectifying channel which we call KGP (or hpKir3.4), to signify that it is K+ selective, G-protein-gated and isolated from human pancreas. KGP expression in Xenopus oocytes resulted in sizeable basal (or agonist-independent) currents while coexpression with a G-protein-linked receptor, yielded additional agonist-induced currents. Coexpression of KGP and hGIRK1 (a human brain homolog of GIRK1/Kir3.1) produced much larger basal currents than those observed with KGP or hGIRK1 alone, and upon coexpression with receptor, similarly large agonist-induced currents could be obtained. Pertussis toxin treatment significantly diminished agonist-dependent currents due to either KGP or KGP/hGIRK1 expression. Interestingly, PTX also significantly reduced basal KGP or KGP/hGIRK1 currents, suggesting that basal activity is largely the result of G-protein gating as well. When the two channels were coexpressed with receptor, the relative increase in current elicited by agonist was similar whether KGP and hGIRK1 were expressed alone or together. When in vitro translated or when expressed in Xenopus oocytes or CHO mammalian cells, KGP gave rise to a nonglycosylated 45-kD protein. Antibodies directed against either KGP or hGIRK1 coprecipitated both proteins coexpressed in oocytes, providing evidence for the heteromeric assembly of the two channels and suggesting that the current potentiation seen with coexpression of the two channel subunits is due to specific interactions between them. An endogenous oocyte protein similar in size to KGP was also coprecipitated with hGIRK1.