KChiP2 Stabilizes Kv4 Protein Expression and Cell Surface Retention to Control Cardiac Ito Channel Densities

Abstract

The myocardial transient outward current (Ito) is encoded by voltage-gated potassium (Kv) channel α-subunits of the Kv4 subfamily, together with the cytosolic accessory subunit, KChIP2. Targeted deletion of KChIP2 (KChIP2-/-) or Kv4.2 (Kv4.2-/-) eliminates Ito in adult mouse ventricular myocytes. Heterologous co-expression with KChIP2 increases Kv4.2 current densities and results in a relative shift in Kv4.2 from a perinuclear localization to the cell surface, leading to the suggestion that KChIP2 alleviates retention of assembled Kv4 channels in the endoplasmic reticulum (ER) and promotes forward trafficking. To explore these hypotheses, a putative RXR-type ER-retention motif at residues 35-RKR-37 in Kv4.2 was mutated (Kv4.2AAA), and the functional consequences of this construct on Kv4.2 expression in human embryonic kidney-293 (HEK) cells were explored. Mean ± SEM peak Kv4.2 current densities in cells expressing Kv4.2AAA (316±50 pA/pF) were significantly (p=0.025) higher than in cells expressing wild type Kv4.2 (174 ± 20 pA/pF). Surprisingly, however, adenoviral expression of Kv4.2AAA in Kv4.2-/- myocytes resulted in peak Kv current densities (86±9 pA/pF) that were not significantly different from the peak Kv currents (72±9 pA/pF) in Kv4.2-/- cells infected with wild type Kv4.2. Heterologous expression of a charge-conservative mutant, Kv4.2KKK, in which arginines 35 and 37 were mutated to lysines (Kv4.2KKK), resulted in Kv4.2 currents (172±25 pA/pF) that were indistinguishable from wild type currents, demonstrating that the presence of charged residues in the Kv4.2 N-terminus affects channel gating, not channel trafficking. Biochemical studies revealed no differences in the surface expression of the Kv4.2AAA mutant and wild type Kv4.2, and the surface expression of both constructs was increased dramatically upon co-expression of KChIP2. The results of further biochemical studies suggest that KChIP2 functions to increase the retention of Kv4.2 channels at the cell surface.