Gating of the large mechanosensitive channel in situ: Estimation of the spatial scale of the transition from channel population responses
Gating of the large mechanosensitive channel in situ: Estimation of the spatial scale of the transition from channel population responses
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DOI：
10.1016/s0006-3495(04)74337-4
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发表时间：
2004-05-01
影响因子：
3.4
通讯作者：
Sukharev, S
中科院分区：
文献类型：
--
作者：
Chiang, CS;Anishkin, A;Sukharev, S
与张力敏通道打开相关的物理扩展与电压门控通道的门控电荷具有相同的含义。尽管越来越多的证据表明MSCL的开放状态构象，但对其复杂门控的能量描述仍然不完整。先前估计的MSCL的平面内扩展比分子模型预测的要小得多。为了解决这一差异，我们对野生型MSCL在大肠杆菌巨球体内的电流和剂量-反应曲线进行了系统的研究。利用全点直方图方法，并对每个贴片中的小机械敏感通道(MSCs)的阈值进行张力校准，我们发现亚导电态之间的通道分布对张力的依赖程度明显低于闭合态和导电态之间的分布。在-20 mV时，所有亚态合计占据了大约30%的打开时间，平均积分电流减少了大约6%，基本上与张力或P-O无关。这与门控方案是一致的，其中主要的限速步骤是在闭合状态和低电导亚态之间的转变，并验证了积分电流作为P-O测量的使用，以及在两态近似下剂量-效应曲线的处理。从29条独立的剂量-反应曲线中提取的DeltaE和DeltaA状态之间的表观能量和面积差以线性相关的方式变化，而中点张力保持在类似于10.4mN/m的水平。统计模拟表明，每个斑块中的通道之间的门控参数略有差异，导致表观DE和DA的显著减少和相关扩散。激活曲线的起始部分的斜率(有几个通道是活跃的)给出了DeltaE=51+/-13kT和DeltaA=20.4+/-4.8 nm(2)，后者与预测DeltaA=23 nm(2)的MSCL的结构模型是一致的。
Physical expansion associated with the opening of a tension-sensitive channel has the same meaning as gating charge for a voltage-gated channel. Despite increasing evidence for the open-state conformation of MscL, the energetic description of its complex gating remains incomplete. The previously estimated in-plane expansion of MscL is considerably smaller than predicted by molecular models. To resolve this discrepancy, we conducted a systematic study of currents and dose-response curves for wild-type MscL in Escherichia coli giant spheroplasts. Using the all-point histogram method and calibrating tension against the threshold for the small mechanosensitive channel (MscS) in each patch, we found that the distribution of channels among the subconducting states is significantly less dependent on tension than the distribution between the closed and conducting states. At -20 mV, all substates together occupy similar to30% of the open time and reduce the mean integral current by similar to6%, essentially independent of tension or P-o. This is consistent with the gating scheme in which the major rate-limiting step is the transition between the closed state and a low-conducting substate, and validates both the use of the integral current as a measure of P-o, and treatment of dose-response curves in the two-state approximation. The apparent energy and area differences between the states DeltaE and DeltaA, extracted from 29 independent dose-response curves, varied in a linearly correlated manner whereas the midpoint tension stayed a similar to10.4 mN/m. Statistical modeling suggests slight variability of gating parameters among channels in each patch, causing a strong reduction and correlated spread of apparent DE and DA. The slope of initial parts of activation curves, with a few channels being active, gave estimates of DeltaE = 51 +/- 13 kT and DeltaA = 20.4 +/- 4.8 nm(2), the latter being consistent with structural models of MscL, which predict DeltaA = 23 nm(2).