The steroid interaction site in transmembrane domain 2 of the large conductance, voltage- and calcium-gated potassium (BK) channel accessory β1 subunit
The steroid interaction site in transmembrane domain 2 of the large conductance, voltage- and calcium-gated potassium (BK) channel accessory β1 subunit
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DOI：
10.1073/pnas.1112901108
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发表时间：
2011-12-13
影响因子：
11.1
通讯作者：
Dopico, Alejandro M.
中科院分区：
文献类型：
--
作者：
Bukiya, Anna N.;Singh, Aditya K.;Dopico, Alejandro M.
大电导、电压门控和钙门控钾（BK）通道调节几种生理过程，包括肌源性张力和动脉直径。越来越多的人认识到类固醇对BK活性的非基因组调节，但类固醇作用的精确位置仍然未知。我们已经证明，石胆酸盐（LC）和相关胆烷类固醇引起的动脉扩张是由血管肌细胞BK活性（EC（50）= 45 μ M）增加2倍引起的，这种作用需要β 1，而不需要其他（β 2-β 4）BK辅助亚基。结合突变和膜片钳在生理条件下的钙和电压对BK α-（cbv 1）和β 1亚基从大鼠脑动脉肌细胞，我们确定了类固醇相互作用的网站从两个区域BK β 1跨膜结构域2提出的计算动力学：外部位点包括L157、L158和T165，而内部位点包括T169、L172和L173。正如计算模型所预期的，cbv 1 +r β 1 T165 A，T169 A通道是LC无反应的。而cbv 1 + r β 1 T165 A和cbv 1 + r β 1 T165 A、L157 A、L158 A对LC完全敏感。数据表明，跨膜结构域2外部网站不有助于类固醇作用。Cbv 1 + r β 1 T169 A对LC不敏感，r β 1 T169 S不能挽救对LC的反应性。此外，cbv 1 + r β 1 L172 A和cbv 1 + r β 1 L173 A通道是LC不敏感的。这些数据和计算模型表明，T169和类固醇α-羟基之间的紧密氢键，以及L172，L173和类固醇环之间的疏水相互作用都是LC作用所必需的。因此，β 1 TM 2 T169、L172、L173为BK通道的胆烷类固醇激活提供了相互作用区域。由于这种氨基酸三联体是BK β 1所独有的，我们的研究为推进BK通道β 1亚基特异性药理学提供了结构基础。
Large conductance, voltage-and calcium-gated potassium (BK) channels regulate several physiological processes, including myogenic tone and thus, artery diameter. Nongenomic modulation of BK activity by steroids is increasingly recognized, but the precise location of steroid action remains unknown. We have shown that artery dilation by lithocholate (LC) and related cholane steroids is caused by a 2x increase in vascular myocyte BK activity (EC(50) = 45 mu M), an action that requires beta 1 but not other (beta 2-beta 4) BK accessory subunits. Combining mutagenesis and patch-clamping under physiological conditions of calcium and voltage on BK alpha-(cbv1) and beta 1 subunits from rat cerebral artery myocytes, we identify the steroid interaction site from two regions in BK beta 1 transmembrane domain 2 proposed by computational dynamics: the outer site includes L157, L158, and T165, whereas the inner site includes T169, L172, and L173. As expected from computational modeling, cbv1+r beta 1T165A, T169A channels were LC-unresponsive. However, cbv1 + r beta 1T165A and cbv1 + r beta 1T165A, L157A, L158A were fully sensitive to LC. Data indicate that the transmembrane domain 2 outer site does not contribute to steroid action. Cbv1 + r beta 1T169A was LC-insensitive, with r beta 1T169S being unable to rescue responsiveness to LC. Moreover, cbv1 + r beta 1L172A, and cbv1 + r beta 1L173A channels were LC-insensitive. These data and computational modeling indicate that tight hydrogen bonding between T169 and the steroid alpha-hydroxyl, and hydrophobic interactions between L172, L173 and the steroid rings are both necessary for LC action. Therefore, beta 1 TM2 T169, L172, L173 provides the interaction area for cholane steroid activation of BK channels. Because this amino acid triplet is unique to BK beta 1, our study provides a structural basis for advancing beta 1 subunit-specific pharmacology of BK channels.