Voltage-gated sodium channels (NaV) initiate the upstroke of the action potential in excitable cells. Recent data highlighted that pH can influence interactions between Ca 2+ -binding proteins and ion channels. Here, we investigated intracellular pH (pH i ) modification of the cardiac sodium channel (Na V 1.5) and accessory protein calmodulin (CaM). Point mutations (H1915A, H1915Y, and H1915K) were used to test intermolecular forces between CaM and Na V 1.5 H1915 at pH 7.4 and 6.6 in the presence and absence of calcium. Fluorescence spectroscopy data demonstrated that CaM-IQ complex Ca 2+ -binding (affinity and cooperativity) was sensitive to pH. Mutations to H1915 were able to minimize or remove this pH effect. ITC was used to investigate the effect of pH on CaM-IQ binding affinity (protein-protein interaction). Thermodynamic properties of H1915A and H1915K (enthalpy and entropy) were pH sensitive. Conversely, CaM-IQ binding affinity was not pH sensitive irrespective of point mutation. NMR spectroscopy data demonstrated that pH altered all CaM-IQ motif interactions. In the presence of Ca 2+ at pH 6.6, removal of the H1915 side chain (H1915A) altered spectra in a manner consistent with impaired complex stability. Whole-cell patch clamp characterized the influence of pH i on Na V function. WT Na V 1.5 was not sensitive to changes in pH i . Intriguingly, H1915A and H1915K Na V 1.5 displayed alterations in function from pH i 7.4 to 6.6. Together, these results highlight the importance of H1915 for appropriate function of this CaM-IQ complex.
Agee et al. (Sun,) studied this question.
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