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.
Keywords: bestrophin; calcium binding; chloride channels; pH dependence; protein electrostatics.