B-Crystallin Interacts with Nav1.5 and Regulates Ubiquitination and Internalization of Cell Surface Nav1.5

Na(v)1.5, the pore-forming subunit of the cardiac voltage-gated Na+ channel complex, is required for the initiation and propagation of the cardiac action potential. Mutations in Na(v)1.5 cause cardiac arrhythmias and sudden death. The cardiac Na+ channel functions as a protein complex; however, its complete components remain to be fully elucidated. A yeast two-hybrid screen identified a new candidate Na(v)1.5-interacting protein, B-crystallin. GST pull-down, co-immunoprecipitation, and immunostaining analyses validated the interaction between Na(v)1.5 and B-crystallin. Whole-cell patch clamping showed that overexpression of B-crystallin significantly increased peak sodium current (I-Na) density, and the underlying molecular mechanism is the increased cell surface expression level of Na(v)1.5 via reduced internalization of cell surface Na(v)1.5 and ubiquitination of Na(v)1.5. Knock-out of B-crystallin expression significantly decreased the cell surface expression level of Na(v)1.5. Co-immunoprecipitation analysis showed that B-crystallin interacted with Nedd4-2; however, a catalytically inactive Nedd4-2-C801S mutant impaired the interaction and abolished the up-regulation of I-Na by B-crystallin. Na(v)1.5 mutation V1980A at the interaction site for Nedd4-2 eliminated the effect of B-crystallin on reduction of Na(v)1.5 ubiquitination and increases of I-Na density. Two disease-causing mutations in B-crystallin, R109H and R151X (nonsense mutation), eliminated the effect of B-crystallin on I-Na. This study identifies B-crystallin as a new binding partner for Na(v)1.5. B-Crystallin interacts with Na(v)1.5 and increases I-Na by modulating the expression level and internalization of cell surface Na(v)1.5 and ubiquitination of Na(v)1.5, which requires the protein-protein interactions between B-crystallin and Na(v)1.5 and between B-crystallin and functionally active Nedd4-2.