Competition of calcified calmodulin N lobe and PIP2to an LQT mutation site in Kv7.1 channel

Abstract

Voltage-gated potassium 7.1 (Kv7.1) channel and KCNE1 protein coassembly forms the slow potassium current IKSthat repolarizes the cardiac action potential. The physiological importance of the IKSchannel is underscored by the existence of mutations in humanKv7.1andKCNE1genes, which cause cardiac arrhythmias, such as the long-QT syndrome (LQT) and atrial fibrillation. The proximal Kv7.1 C terminus (CT) binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP2), but the role of CaM in channel function is still unclear, and its possible interaction with PIP2is unknown. Our recent crystallographic study showed that CaM embraces helices A and B with the apo C lobe and calcified N lobe, respectively. Here, we reveal the competition of PIP2and the calcified CaM N lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor an LQT mutation. Protein pulldown, molecular docking, molecular dynamics simulations, and patch-clamp recordings indicate that residues K526 and K527 in Kv7.1 helix B form a critical site where CaM competes with PIP2to stabilize the channel open state. Data indicate that both PIP2and Ca2+-CaM perform the same function on IKSchannel gating by producing a left shift in the voltage dependence of activation. The LQT mutant K526E revealed a severely impaired channel function with a right shift in the voltage dependence of activation, a reduced current density, and insensitivity to gating modulation by Ca2+-CaM. The results suggest that, after receptor-mediated PIP2depletion and increased cytosolic Ca2+, calcified CaM N lobe interacts with helix B in place of PIP2to limit excessive IKScurrent inhibition.