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

2016; Elsevier BV; Volume: 110; Issue: 3 Linguagem: Inglês

10.1016/j.bpj.2015.11.1037

1542-0086

William S. Tobelaim, Meidan Dvir, Guy Lebel, Meng Cui, Tal Buki, Asher Peretz, Diomedes E. Logothetis, Joel A. Hirsch, Bernard Attali,

Ion channel regulation and function

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.