Two novel Brugada syndrome-associated mutations increase K_V4.3 membrane expression and function

The human cardiac fast transient outward K⁺ channel is composed of the K_V4.3 α subunit encoded by KCND3 and the K⁺ channel-interacting protein 2 (KChIP2) β subunit, and determines the early repolarization of the action potential (AP). Two human mutations (G600R and L450F) in K_V4.3 are associated with Brugada syndrome and they increase the K_V4.3/KChIP2-encoded fast transient outward K⁺ current (I_to,f) and cause the stable loss of the AP dome. However, the detailed mechanisms underlying the gain of I_to,f function by these two mutations are largely unknown. The experiments in the present study were undertaken to investigate the effect of these mutations and the underlying mechanism. Whole cell patch-clamp recording was performed in HEK-293 cells expressing KV₄.3-wild-type (WT) and K_V4.3 mutants with KChIP2. The two individual mutant-encoded currents were significantly increased but the kinetics of the channels affected by the two mutations were different. The two mutations slowed K_V4.3/KChIP2-encoded channel inactivation; they did not increase the recovery from the K_V4.3/KChIP2-encoded channel inactivation. Western blotting showed that total K_V4.3 protein was significantly augmented in HEK-293 cells expressing the two individual mutants with KChIP2. Furthermore, immunofluorescence confocal microscopy demonstrated that the K_V4.3 channel protein was expressed more in the cell membrane compared to the cytoplasm in cells that expressed individual mutants with KChIP2. Also, KChIP2 increased the amount of channel protein in the cell membrane of K_V4.3 mutants significantly more than K_V4.3-WT. Reverse transcription-polymerase chain reaction showed that K_V4.3 mRNA was not significantly changed by individual mutations in the presence of KChIP2. Taken together, the present study revealed that the mutations cause a gain-of-function of K_V4.3/KChIP2-encoded channels by increasing membrane protein expression and slowing channel inactivation.