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Cytosolic Kv channel-interacting proteins KChIP1-4 co-assemble with pore-forming Kv4 subunits to form a native complex that encodes a somatodendritic A-type K+ current.KChIP4a, a splice variant of KChIP4, shares a high homology with other KChIPs in conserved C-terminal core region, but exhibits a distinct modulation on Kv4 current expression and gating by its unique N terminus that is termed K+ channel inactivation suppressor (KIS) domain.It has been shown that N-terminal KIS of KChIP4a slows inactivation of Kv4 and inhibits current expression.Yet, how the KIS domain of KChIP4a suppresses Kv4 current and affects the channel gating remains unknown.In this study, we identified a hydrophobic ER-retention motif within the KIS domain of KChIP4a that suppresses Kv4 surface expression using confocal imaging and cell surface biotinylation assay.The ER retention of KIS domain overwrites its core mediated enhancement of Kv4 surface expression, leading to an outcome of current reduction.Further dissection of KIS domain revealed several key residues that cause reduction of Kv4.3 peak current, but not affect surface expression.Examination of activation and inactivation properties of Kv4.3 co-expressed with either KChIP4a or its core domain (without KIS) demonstrated that 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.Therefore, we propose that KIS domain of KChIP4a inhibits Kv4 function through dual independent mechanisms mediated by ER retention and enhancement of channel closed-state inactivation.