Partially active channels produced by PKA site mutation of the cloned renal K+ channel, ROMK2 (kir1.2).
Partially active channels produced by PKA site mutation of the cloned renal K+ channel, ROMK2 (kir1.2).
复制标题
由克隆肾 K 通道 ROMK2 (kir1.2) 的 PKA 位点突变产生的部分活性通道。
DOI：
10.1152/ajprenal.1998.275.3.f415
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Hebert,SC
中科院分区：
文献类型：
--
作者：
MacGregor,GG;Xu,JZ;McNicholas,CM;Giebisch,G;Hebert,SC
克隆的肾K+通道（ROMK 2）的活性依赖于磷酸化和去磷酸化之间的平衡。ROMK 2上只有三个蛋白激酶A（PKA）位点，磷酸化残基为丝氨酸-25（S25）、丝氨酸-200（S200）和丝氨酸-294（S294）（Z. C. Xu，Y. Yang和S. C. Hebert.J.Biol.Chem.271：9313-9319，1996）。我们先前将这些位点从丝氨酸突变为丙氨酸，以研究每个位点对整体通道功能的贡献。在这里，我们研究了这些单PKA位点突变体使用单通道配置的膜片钳技术。与野生型ROMK 2相比，S200 A和S294 A位点的COOH末端突变均显示出降低的开放通道概率（Po），而S25 A位点的NH 2末端突变显示Po没有变化。S200 A和S294 A突变体的inPo降低是由于长封闭状态的额外存在引起的。相比之下，S25 A通道的出现少了1.66%，表明膜上的活性通道较少。与野生型ROMK 2通道相比，S200 A和S294 A通道具有不同的动力学，显示亚水平的发生增加。当野生型ROMK 2被切除并暴露于去磷酸化条件时，观察到类似的动力学，表明这些效应是部分磷酸化通道的特异性特性，而不是由于突变的无关效应。
The activity of the cloned renal K+channel (ROMK2) is dependent on a balance between phosphorylation and dephosphorylation. There are only three protein kinase A (PKA) sites on ROMK2, with the phosphorylated residues being serine-25 (S25), serine-200 (S200), and serine-294 (S294) (Z.-C. Xu, Y. Yang, and S. C. Hebert.J. Biol. Chem.271: 9313–9319, 1996). We previously mutated these sites from serine to alanine to study the contribution of each site to overall channel function. Here we have studied each of these single PKA site mutants using the single-channel configuration of the patch-clamp technique. Both COOH-terminal mutations at sites S200A and S294A showed a decreased open channel probability (Po), whereas the NH2-terminal mutation at site S25A showed no change inPocompared with wild-type ROMK2. The decrease inPofor the S200A and S294A mutants was caused by the additional presence of a long closed state. In contrast, the occurrence of the S25A channel was ∼66% less, suggesting fewer active channels at the membrane. The S200A and S294A channels had different kinetics compared with wild-type ROMK2 channels, showing an increased occurrence of sublevels. Similar kinetics were observed when wild-type ROMK2 was excised and exposed to dephosphorylating conditions, indicating that these effects are specifically a property of the partially phosphorylated channel and not due to an unrelated effect of the mutation.