The mechanism of fast-gate opening in ClC-0
The mechanism of fast-gate opening in ClC-0
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DOI：
10.1085/jgp.200709759
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发表时间：
2007-10-01
影响因子：
3.8
通讯作者：
Maduke, Merritt
中科院分区：
文献类型：
--
作者：
Engh, Anita M.;Faraldo-Gomez, Jose D.;Maduke, Merritt
ClC-0是一种氯离子通道，其门控对电压和氯离子都敏感。基于使用单通道记录的门控动力学分析，提出了五态模型来描述ClC-0快速门控打开对电压和外部氯化物的依赖性（Chen，T.是的，和C.米勒。1996. 108：237-250）。我们的目标是使用这个五态模型作为理解门控过程中发生的结构变化的起点。使用宏观补丁记录，我们能够重现电压和氯化物的影响，陈和米勒报告，并适合我们的开放速率常数数据的五态模型。在进一步分析我们的数据以及Chen和米勒的数据后，我们了解到与他们的结论相反，（a）数据中的特征不足以排除更简单的四态模型，（B）氯离子结合步骤是电压依赖性的。为了能够评估突变体对门控的影响（在配套论文中描述，参见Engh et al. on p. 351 of this issue），我们开发了一种确定门控模型参数误差的方法，并评估了该误差的来源。为了开始将动力学模型与已知的CLC结构相结合，基于原核同源物ClC-ec 1的X射线晶体结构计算生成ClC-0的模型。在该同源模型中的孔静电分析表明，从门控动力学分析得出的结论中至少有两个与已知的CLC结构一致：（1）氯离子结合是通道开放所必需的，以及（2）氯离子与三个已知氯离子结合位点中的任何一个的结合必须是电压依赖性的。
ClC-0 is a chloride channel whose gating is sensitive to both voltage and chloride. Based on analysis of gating kinetics using single-channel recordings, a five-state model was proposed to describe the dependence of ClC-0 fast-gate opening on voltage and external chloride (Chen, T.-Y., and C. Miller. 1996. J. Gen. Physiol. 108: 237-250). We aimed to use this five-state model as a starting point for understanding the structural changes that occur during gating. Using macroscopic patch recordings, we were able to reproduce the effects of voltage and chloride that were reported by Chen and Miller and to fit our opening rate constant data to the five-state model. Upon further analysis of both our data and those of Chen and Miller, we learned that in contrast to their conclusions, (a) the features in the data are not adequate to rule out a simpler four-state model, and (b) the chloride-binding step is voltage dependent. In order to be able to evaluate the effects of mutants on gating (described in the companion paper, see Engh et al. on p. 351 of this issue), we developed a method for determining the error on gating model parameters, and evaluated the sources of this error. To begin to mesh the kinetic model(s) with the known CLC structures, a model of ClC-0 was generated computationally based on the X-ray crystal structure of the prokaryotic homolog ClC-ec1. Analysis of pore electrostatics in this homology model suggests that at least two of the conclusions derived from the gating kinetics analysis are consistent with the known CLC structures: (1) chloride binding is necessary for channel opening, and (2) chloride binding to any of the three known chloride-binding sites must be voltage dependent.