Enhancement of Closed-State Inactivation and ER Retention of Kv4.3 Mediated by N-Terminal KIS Domain of Auxiliary KChIP4A

Abstract

Auxiliary KChIP4a shares high homology of conserved C-terminal core region with other members of Kv channel-interacting proteins (KChIPs), but exhibits a distinct modulation on Kv4 current expression and gating. It has been shown that the unique N-terminus of KChIP4a functions as K+ channel inactivation suppressor (KIS) that leads to slow inactivation and current inhibition. However, the mechanism by which the KIS domain causes current reduction remains unknown. In this study, we identified a hydrophobic ER-retention motif within the KIS domain of KChIP4a that suppresses Kv4.3 surface expression using confocal imaging and cell surface biotinylation assay. Further dissection of KIS domain revealed several key residues that cause reduction of Kv4.3 peak current, but do not affect surface expression. Examination of gating properties of Kv4.3 co-expressed with either KChIP4a or its core without the KIS (KChIP4aΔ2-34) demonstrated that KChIP4a with KIS domain had no significant effect on steady-state activation, but shifted the voltage dependence of steady-state inactivation of Kv4.3 to hyperpolarizing direction by enhancing closed-state inactivation. We have previously demonstrated that KChIP4a can rescue the function of a tetramerization-defect mutant Kv4.3 C110A. The rescued Kv4.3 C110A current is larger than that of WT Kv4.3/KChIP4a co-expression, although C110A surface expression was lower. Upon coexpression, the closed-state inactivation of Kv4.3 C110A mutant was less affected by the KIS domain, as compared with enhanced closed-state inactivation observed in wild-type channel, suggesting a role of T1 domain in mediation of Kv4 closed-state inactivation. Taken together, we propose that N-terminal KIS domain of KChIP4a inhibits Kv4.3 function through dual independent mechanisms by which auxiliary KChIP4a causes Kv4 ER retention and promotes channel closed-state inactivation.