Mechanism of ATP-sensitive K channel inhibition by sulfhydryl modification
Mechanism of ATP-sensitive K channel inhibition by sulfhydryl modification
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DOI：
10.1085/jgp.112.3.325
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发表时间：
1998-09-01
影响因子：
3.8
通讯作者：
Ashcroft, FM
中科院分区：
文献类型：
--
作者：
Trapp, S;Tucker, SJ;Ashcroft, FM
ATP敏感钾（K-ATP）通道被细胞内ATP可逆地抑制。当作用于细胞膜表面时，与巯基部分相互作用的药物对K-ATP通道活性产生不可逆的抑制作用。ATP似乎可以防止这种影响，这表明与巯基试剂相互作用的半胱氨酸残基可能位于ATP结合位点内，或者当通道关闭时无法进入。我们已经研究了膜-不含硫醇反应剂对氯甲基苯基磺酸（pCMPS）与克隆β细胞K-ATP通道的相互作用。该通道由形成孔隙的Kir6.2和调控的SUR1亚基组成。我们发现参与pCMPS通道抑制的半胱氨酸残基位于Kir6.2亚基上，位于42号位置，位于蛋白质的NH2末端。尽管ATP可以抵抗pCMPS的影响，但K-ATP通道的ATP敏感性不会因C42突变对缬氨酸（CV）或丙氨酸(A)的影响而改变，这表明ATP不会直接与该残基相互作用。这些结果与细胞内溶液无法接触C42的观点一致，因此当通道被ATP关闭时，C42免受pCMPS的相互作用。我们还观察到，C42A突变不影响SUR1赋予Kir6.2二氮氧化物敏感性的能力，并降低但不阻止MgADP和甲苯丁胺的作用，这些作用是通过SUR1介导的。Kir6.2-C42A（或V）突变通道可能为半胱氨酸扫描诱变研究提供合适的背景。
ATP-sensitive potassium (K-ATP) channels are reversibly inhibited by intracellular ATP. Agents that interact with sulfhydryl moieties produce an irreversible inhibition of K-ATP channel activity when applied to the intracellular membrane surface. ATP appears to protect against this effect, suggesting that the cysteine residue with which thiol reagents interact may either lie within the ATP-binding site or be inaccessible when the channel is closed. We have examined the interaction of the membrane-impermeant thiol-reactive agent p-chloromercuriphenyl-sulphonate (pCMPS) with the cloned beta cell K-ATP channel. This channel comprises the pore-forming Kir6.2 and regulatory SUR1 subunits. We show that the cysteine residue involved in channel inhibition by pCMPS resides on the Kir6.2 subunit and is located at position 42, which lies within the NH2 terminus of the protein. Although ATP protects against the effects of pCMPS, the ATP sensitivity of the K-ATP channel nas unchanged by mutation of C42 to either valine CV) or alanine (A), suggesting that ATP does not interact directly with this residue. These results are consistent with the idea that C42 is inaccessible to the intracellular solution, and thereby protected from interaction with pCMPS when the channel is closed by ATP. We also observed that the C42A mutation does not affect the ability of SUR1 to endow Kir6.2 with diazoxide sensitivity, and reduces, but does not prevent, the effects of MgADP and tolbutamide, which are mediated via SUR1. The Kir6.2-C42A (or V) mutant channel may provide a suitable background for cysteine-scanning mutagenesis studies.