Modulation of human Kv4.3/KChIP2 channel inactivation kinetics by cytoplasmic Ca 2

The transient outward current (I ) in the human heart is mediated by Kv4.3 channels complexed with Kv channel interacting protein (KChIP) 2, a cytoplasmic Ca -binding EF-hand protein known to modulate Kv4.3 inactivation gating upon heterologous co-expression. We studied Kv4.3 channels co-expressed with wild-type (wt) or EF-hand-mutated (ΔEF) KChIP2 in human embryonic kidney (HEK) 293 cells. Co-expression took place in the absence or presence of BAPTA-AM, and macroscopic currents were recorded in the whole-cell patch-clamp configuration with different free Ca concentrations in the patch-pipette. Our data indicate that Ca is not necessary for Kv4.3/KChIP2 complex formation. The Kv4.3/KChIP2-mediated current decay was faster and the recovery of Kv4.3/KChIP2 channels from inactivation slower with 50 μM Ca than with BAPTA (nominal Ca -free) in the patch-pipette. The apparent Ca -mediated slowing of recovery kinetics was still observed when EF-hand 4 of KChIP2 was mutated (ΔEF4) but not when EF-hand 2 (ΔEF2) was mutated, and turned into a Ca -mediated acceleration of recovery kinetics when EF-hand 3 (ΔEF3) was mutated. In the presence of the Ca /calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 cytoplasmic Ca (50 μM) induced an acceleration of Kv4.3/KChIP2 recovery kinetics, which was still observed when EF-hand 2 was mutated (ΔEF2) but not when EF-hand 3 (ΔEF3) or EF-hand 4 (ΔEF4) was mutated. Our results support the notion that binding of Ca to KChIP2 EF-hands can acutely modulate Kv4.3/KChIP2 channel inactivation gating, but the Ca -dependent gating modulation depends on CaMKII action. Our findings speak for an acute modulation of I kinetics and frequency-dependent I availability in cardiomyocytes under conditions with elevated Ca levels and CaMKII activity.