Computational investigation of mechanisms for pH modulation of human chloride channels
Computational investigation of mechanisms for pH modulation of human chloride channels
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人氯通道 pH 调节机制的计算研究
DOI：
10.1101/2022.10.03.510624
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Elverson K
中科院分区：
文献类型：
--
作者：
Elverson K
许多跨膜蛋白都受细胞内或细胞外pH的调节。PH依赖性的研究通常是通过一系列氨基酸的突变来进行的，受氨基酸保守和结构等性质的指导。预测pKas可以简化这一过程，允许快速和有效地识别与pH相关的感兴趣的氨基酸。从钙激活的氯离子通道Bestrophin 1开始，在没有钙的情况下，钙位点周围的羧酸配体结构松弛，这与测量到的缺乏pH依赖性是一致的。相反，建议在TMEM16A中无钙时较少松弛，并维持升高的羧酸侧链pKas，以产生pH依赖的氯通道活动。这一假说通过结构松弛程度来调节钙/质子偶联和pH依赖的活性，这一假说被证明适用于特征良好的胞浆蛋白CaM(pH无关)和Calbindin D9k(pH依赖)。不稳定的、可电离的电荷位置的进一步应用，或静电受挫，被应用于其他人氯通道(不是钙激活的)、ClC-2、GABAA和GlyR。通过实验确定的pH调节位置很容易确定。基于结构的PKA预测工具是免费提供的，允许用户专注于突变研究，构建假想的质子途径，并推导出假说，如通过结构灵活性控制pH依赖的钙激活的模型。预测离子通道紊乱突变的pH依赖关系可以支持实验，并最终支持临床干预。
Many transmembrane proteins are modulated by intracellular or extracellular pH. Investigation of pH dependence generally proceeds by mutagenesis of a wide set of amino acids, guided by properties such as amino-acid conservation and structure. Prediction of pKas can streamline this process, allowing rapid and effective identification of amino acids of interest with respect to pH dependence. Commencing with the calcium-activated chloride channel bestrophin 1, the carboxylate ligand structure around calcium sites relaxes in the absence of calcium, consistent with a measured lack of pH dependence. By contrast, less relaxation in the absence of calcium in TMEM16A, and maintenance of elevated carboxylate sidechain pKas, is suggested to give rise to pH-dependent chloride channel activity. This hypothesis, modulation of calcium/proton coupling and pH-dependent activity through the extent of structural relaxation, is shown to apply to the well-characterised cytosolic proteins calmodulin (pH-independent) and calbindin D9k (pH-dependent). Further application of destabilised, ionisable charge sites, or electrostatic frustration, is made to other human chloride channels (that are not calcium-activated), ClC-2, GABAA, and GlyR. Experimentally determined sites of pH modulation are readily identified. Structure-based tools for pKa prediction are freely available, allowing users to focus on mutagenesis studies, construct hypothetical proton pathways, and derive hypotheses such as the model for control of pH-dependent calcium activation through structural flexibility. Predicting altered pH dependence for mutations in ion channel disorders can support experimentation and, ultimately, clinical intervention.