Computational investigation of mechanisms for pH modulation of human chloride channels

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. This study shows how web-based calculation of pKas allows 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 extent of structural relaxation, is shown to apply to the well-characterised cytosolic proteins calmodulin (pH-independent) and calbindin D 9k (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, GABA A , and GlyR. Experimentally-determined sites of pH modulation are readily identified. The structure-based tool is freely available, allowing users to focus 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.