Cysteine mutagenesis and computer modeling of the S6 region of an intermediate conductance IKCa channel.
Cysteine mutagenesis and computer modeling of the S6 region of an intermediate conductance IKCa channel.
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DOI：
10.1085/jgp.20028586
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发表时间：
2002-07
期刊：
影响因子：
--
通讯作者：
Sauvé R
中科院分区：
文献类型：
--
作者：
Simoes M;Garneau L;Klein H;Banderali U;Hobeila F;Roux B;Parent L;Sauvé R
半胱氨酸扫描诱变（SCAM）和基于计算机的建模被用来调查的S6跨膜段的钙激活的K+通道的中间电导IKCa的关键结构特征。我们的SCAM结果表明，[2-（三甲基铵）乙基]甲硫基磺酸溴化物（MTSET）与在位置275，278和282处工程化的半胱氨酸的相互作用导致电流抑制。这种效应是状态依赖性的，因为MTSET在封闭状态（零Ca 2+条件）下抑制IKCa的效果不如开放状态配置。我们的研究结果还表明，在开放的IKCa通道中，S6的最后四个残基，从A283到A286，完全暴露于水，而MTSET仍然可以到达283 C和286 C残基，IKCa保持在封闭状态构型。值得注意的是，内部应用MTSET或（2-磺酰乙基）甲硫基磺酸钠（MTSES）引起了强烈的Ca 2+依赖性刺激的A283 C，V285 C和A286 C电流。然而，与野生型IKCa相反，MTSET刺激的A283 C和A286 C电流似乎是TEA不敏感的，表明在位置283和286处的MTSET结合损害了TEA进入通道孔。接下来使用KcsA结构作为模板通过同源建模生成三维结构数据。根据SCAM结果，三维模型预测V275、T278和V282残基应该衬在通道孔中。然而，A283-A286区域的孔尺寸不能解释MTSET对封闭A283 C和A286突变体的影响。我们的研究结果表明，从V275延伸到V282的S6域具有相应的KcsA的内腔区域的功能，和COOH末端的S6，从A283到A286，是更灵活的比单独的封闭的KcsA晶体结构的基础上预测。根据该模型，门的关闭应该发生在位于T278和V282残基之间的点处。
Cysteine-scanning mutagenesis (SCAM) and computer-based modeling were used to investigate key structural features of the S6 transmembrane segment of the calcium-activated K+ channel of intermediate conductance IKCa. Our SCAM results show that the interaction of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) with cysteines engineered at positions 275, 278, and 282 leads to current inhibition. This effect was state dependent as MTSET appeared less effective at inhibiting IKCa in the closed (zero Ca2+ conditions) than open state configuration. Our results also indicate that the last four residues in S6, from A283 to A286, are entirely exposed to water in open IKCa channels, whereas MTSET can still reach the 283C and 286C residues with IKCa maintained in a closed state configuration. Notably, the internal application of MTSET or sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) caused a strong Ca2+-dependent stimulation of the A283C, V285C, and A286C currents. However, in contrast to the wild-type IKCa, the MTSET-stimulated A283C and A286C currents appeared to be TEA insensitive, indicating that the MTSET binding at positions 283 and 286 impaired the access of TEA to the channel pore. Three-dimensional structural data were next generated through homology modeling using the KcsA structure as template. In accordance with the SCAM results, the three-dimensional models predict that the V275, T278, and V282 residues should be lining the channel pore. However, the pore dimensions derived for the A283–A286 region cannot account for the MTSET effect on the closed A283C and A286 mutants. Our results suggest that the S6 domain extending from V275 to V282 possesses features corresponding to the inner cavity region of KcsA, and that the COOH terminus end of S6, from A283 to A286, is more flexible than predicted on the basis of the closed KcsA crystallographic structure alone. According to this model, closure by the gate should occur at a point located between the T278 and V282 residues.
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影响因子：
56.9
作者：
Doyle, DA;Cabral, JM;MacKinnon, R
通讯作者：
MacKinnon, R
影响因子：
2.4
作者：
Dunn, PM
通讯作者：
Dunn, PM
影响因子：
3.4
作者：
MORIER, N;SAUVE, R
通讯作者：
SAUVE, R
影响因子：
4.8
作者：
Logsdon, NJ;Kang, JS;Aiyar, J
通讯作者：
Aiyar, J
影响因子：
3
作者：
BROOKS, BR;BRUCCOLERI, RE;KARPLUS, M
通讯作者：
KARPLUS, M