Abstract 468: Identification of a New Component of the Na v 1.5 Complex: αB-crystallin Interacts with Na v 1.5 and Regulates Ubiquitination and Internalization of Cell Surface Na v 1.5

Abstract

Mutations in cardiac Na + channel 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, αB-crystallin. GST-pull-down, co-immunoprecipitation and immunostaining analyses validated the interaction between Na v 1.5 and αB-crystallin. Overexpression of αB-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. Knockout of αB-crystallin expression significantly decreased the cell-surface expression level of Na v 1.5. αB-crystallin interacted with Nedd4-2, however, a catalytically inactive Nedd4-2-C801S mutant reduced the interaction between αB-crystallin and Nedd4-2 and also blocked the increase of peak I Na density by αB-crystallin. Na v 1.5 mutation V1980A at the interaction site for Nedd4-2 eliminated the effect of αB-crystallin on reduction of Na v 1.5 ubiquitination and increases of I Na density. The data suggest that the interactions between αB-crystallin and functionally active Nedd4-2 and between αB-crystallin and Na v 1.5 are critical to increased I Na density by αB-crystallin. Multiple mutations in αB-crystallin have been associated with human diseases. Two mutations, R109H and R151X, eliminated the effect of αB-crystallin on I Na . This study identifies αB-crystallin as a new binding partner for Na v 1.5. αB-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 αB-crystallin and Na v 1.5 and between αB-crystallin and functionally active Nedd4-2.