General anesthetic action at an internal protein site involving the S4-S5 cytoplasmic loop of a neuronal K+ channel
General anesthetic action at an internal protein site involving the S4-S5 cytoplasmic loop of a neuronal K+ channel
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DOI：
10.1074/jbc.275.7.4928
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发表时间：
2000-02-18
影响因子：
4.8
通讯作者：
Covarrubias, M
中科院分区：
文献类型：
--
作者：
Harris, T;Shahidullah, M;Covarrubias, M
使用一系列的同源l-烷醇，电生理学，和突变分析的结构基础的全身麻醉剂对神经元K+通道的作用进行了研究。dShaw 2之间的域交换（烷醇敏感）和hKv3.4（链烷醇抗性）和定点诱变证明，13个氨基酸的胞质环（S4-S5）决定了1-链烷醇对天然dShaw 2通道的选择性抑制。S4-S5环可能有助于1-链烷醇和失活颗粒两者的受体，因为携带S4-S5突变的hKv3.4通道的增强的1-烷醇敏感性与被破坏的通道失活直接相关。一个离散的蛋白质位点的证据也从效力和烷基链长度之间的关系的分析，1-己醇后开始趋于平稳。快速应用于由内而外的膜贴片的细胞质侧表明，当药物外部应用于由外而外的膜贴片时，dShaw 2通道和l-烷醇之间的相互作用以慢1000倍的速度平衡。这些数据强烈支持的抑制机制，涉及一个离散的内部网站的l-烷醇dShaw 2 K+通道。一个新的工作假说提出，l-烷醇锁定dShaw 2通道在其封闭的构象，通过直接相互作用在由S4-S5环形成的缝隙。
The structural bases of general anesthetic action on a neuronal K+ channel were investigated using the series of homologous l-alkanols, electrophysiology, and mutational analysis. Domain swapping between dShaw2 (alkanol-sensitive) and hKv3.4 (alkanol-resistant) and site-directed mutagenesis demonstrated that a 13-amino acid cytoplasmic loop (S4-S5) determines the selective inhibition of native dShaw2 channels by l-alkanols, The S4-S5 loop may contribute to a receptor for both l-alkanols and the inactivation particle, because the enhanced l-alkanol sensitivity of hKv3.4 channels hosting S4-S5 mutations correlates directly with disrupted channel inactivation. Evidence of a discrete protein site was also obtained from the analysis of the relationship between potency and alkyl chain length, which begins to level off after 1-hexanol. Rapid application to the cytoplasmic side of inside-out membrane patches shows that the interaction between dShaw2 channels and l-alkanols equilibrates in 1000 fold slower when the drug is applied externally to outside-out membrane patches. The data strongly favor a mechanism of inhibition involving a discrete internal site for l-alkanols in dShaw2 K+ channels. A new working hypothesis proposes that l-alkanols lock dShaw2 channels in their closed conformation by a direct interaction at a crevice formed by the S4-S5 loop.