A Novel Site of Competitive PIP2 and Calmodulin Interaction to KCNQ1 C-Terminus Helix B is Crucial for IKs Channel Activity

Abstract

KCNQ1 and KCNE1 co-assembly generates the IKS potassium current, which is crucial to the cardiac action potential repolarization. Mutations in their corresponding genes cause cardiac arrhythmias. In the KCNQ1 C-terminus (CT), proximal helices A and B form sites for calmodulin (CaM) binding, whereas distal coiled-coil helices C and D are necessary for subunit tetramerization. Studies identified basic residues in KCNQ1 at S2-S3 and S4-S5 intracellular linkers and proximal CT as PIP2 binding sites. Our recent crystallographic data showed that CaM embraces the two proximal anti-parallel helices B and A with its calcified N-lobe and apo C-lobe, respectively. Here we identified a novel site of competitive PIP2 and calmodulin interaction to helix B that is crucial for IKS channel activity. PIP2 competes with CaM binding to purified His-tagged KCNQ1 CT in the presence of Ca2+ only (IC50 = 39 µM). Conversely, recombinant WT Ca2+-CaM or CaM3,4 (IC50 = 1 µM) but neither WT apoCaM nor CaM1,2 compete with PIP2 binding to purified His-tagged KCNQ1 CT. Recombinant WT Ca2+-CaM or CaM3,4 but not WT apoCaM or CaM1,2 in the patch pipet significantly attenuate the decrease in IKS current resulting from PIP2 breakdown by Dr-VSP. Molecular docking, dynamic simulation and subsequent biochemical and electrophysiological validation experiments indicate that K526 and K527 in helix B form a novel and crucial site for competitive PIP2 and calmodulin interaction. Our data suggest that upon PIP2 breakdown (e.g. following GPCR-Gq signaling), PIP2 unbinds from helix B and allows the calcified CaM N-lobe to replace it, which guaranties the maintenance of the channel open state in front of stressful PIP2 depletion events.