Key result
Computational prediction of pKas and structural relaxation successfully identified mechanisms of pH-dependent calcium activation and electrostatic frustration in human chloride channels.
Why the study?
Many transmembrane proteins are modulated by pH, and predicting pKas can streamline mutagenesis studies to identify amino acids of interest governing pH dependence.
Structure-based tools for pKa prediction can identify sites of pH modulation in human chloride channels, supporting mutagenesis studies and hypotheses on pH-dependent activation.
May aid mutagenesis studies of pH-sensitive channels; leaves open translation to human disease models.
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.
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Elverson et al. (2023) studied Ion channel disorders (human chloride channels). Computational pKa prediction and structural analysis was evaluated on Identification of amino acids of interest with respect to pH dependence. Computational prediction of pKas and structural relaxation successfully identified mechanisms of pH-dependent calcium activation and electrostatic frustration in human chloride channels.
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