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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >The steroid interaction site in transmembrane domain 2 of the large conductance, voltage- and calcium-gated potassium (BK) channel accessory SS1 subunit
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The steroid interaction site in transmembrane domain 2 of the large conductance, voltage- and calcium-gated potassium (BK) channel accessory SS1 subunit

机译：大电导，电压门控和钙门控钾（BK）通道附属SS1亚基跨膜结构域2中的类固醇相互作用位点

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摘要

Large conductance, voltage- and calcium-gated potassium (BK) channels regulate several physiological processes, including myo-genic 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 μM), an action that requires p1 but not other (β2-β4) BK accessory subunits. Combining mutagenesis and patch-clamping under physiological conditions of calcium and voltage on BK a- (cbv1) and β1 subunits from rat cerebral artery myocytes, we identify the steroid interaction site from two regions-in BK β1 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+rβ1T165A,T169A channels were LC-unresponsive. However, cbv1 + rβ1T165A and cbv1 + rβ1T165A,L157A,L158A were fully sensitive to LC. Data indicate that the transmembrane domain 2 outer site does not contribute to steroid action. Cbv1 + rβ1T169A was LC-insensitive, with rβ1T169S being unable to rescue responsiveness to LC. Moreover, cbv1 + rβ1L172A, and cbv1 + rβ1L173A channels were LC-insensitive. These data and computational modeling indicate that tight hydrogen bonding between T169 and the steroid a-hydroxyl, and hydrophobic interactions between L172, L173 and the steroid rings are both necessary for LC action. Therefore, β1 TM2 T169,L172,L173 provides the interaction area for cholane steroid activation of BK channels. Because this amino acid triplet is unique to BK β1, our study provides a structural basis for advancing β1 subunit-specific pharmacology of BK channels.
机译：大的电导，电压门控和钙门控钾（BK）通道可调节多种生理过程，包括成肌音，从而调节动脉直径。越来越多地认识到类固醇对BK活性的非基因组调节作用，但类固醇作用的确切位置仍然未知。我们已经表明，通过胆甾醇（LC）和相关的胆甾醇类固醇扩张动脉是由血管肌细胞BK活性增加2倍引起的（EC_（50）= 45μM），这种作用需要p1但不需要其他（β2-β4）BK辅助亚单位。结合诱变和膜片钳在钙和电压的生理条件下对大鼠脑动脉心肌BK a-（cbv1）和β1亚基的诱变和结合，我们通过计算动力学从BKβ1跨膜结构域2的两个区域中识别类固醇相互作用的位点：外部站点包括L157，L158和T165，而内部站点包括T169，L172和L173。如计算模型所预期的那样，cbv1 +rβ1T165A，T169A通道对LC无响应。但是，cbv1 +rβ1T165A和cbv1 +rβ1T165A，L157A，L158A对LC完全敏感。数据表明跨膜结构域2的外部站点不有助于类固醇的作用。 Cbv1 +rβ1T169A对LC不敏感，rβ1T169S无法挽救对LC的反应。此外，cbv1 +rβ1L172A和cbv1 +rβ1L173A通道对LC不敏感。这些数据和计算模型表明，T169与类固醇α-羟基之间的紧密氢键键合以及L172，L173与类固醇环之间的疏水相互作用都是LC作用所必需的。因此，β1TM2 T169，L172，L173为胆甾醇类固醇激活BK通道提供了相互作用区域。由于该氨基酸三联体是BKβ1所特有的，因此我们的研究为推进BK通道的β1亚基特异性药理学提供了结构基础。

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