D242N, a KV7.1 LQTS Mutation Uncovers a KEY Residue for IKS Voltage Dependence

Abstract

Words: 5891Moreno et al., D242N mutation in KV7.1 causes long QT syndrome 1 Abstract KV7.1 and KCNE1 co-assemble to give rise to the IKs current, one of the most important repolarizing currents of the cardiac action potential. Its relevance is underscored by the identification of more than 500 mutations in KV7.1 and, at least, 36 in KCNE1, that cause Long QT Syndrome (LQTS). The aim of this study was to characterize the biophysical and cellular consequences of the D242N KV7.1 mutation associated with the LQTS. The mutation is located in the S4 transmembrane segment, within the voltage sensor of the KV7.1 channel, disrupting the conserved charge balance of this region. Perforated patch-clamp experiments show that, unexpectedly, the mutation did not disrupt the voltage-dependent activation but it removed the inactivation and slowed the activation kinetics of D242N KV7.1 channels. Biotinylation of cell-surface protein and co-immunoprecipitation experiments revealed that neither plasma membrane targeting nor co-assembly between KV7.1 and KCNE1 was altered by the mutation. However, the association of D242N KV7.1 with KCNE1 strongly shifted the voltage dependence of activation to more depolarized potentials (+50mV), hindering IKs current at physiologically relevant membrane potentials. Both functional and computational analysis suggest that the clinical phenotype of the LQTS patients carrying the D242N mutation is due to impaired action potential adaptation to exercise and, in particular, to increase in heart rate. Moreover, our data identify D242 aminoacidic position as a potential residue involved in the KCNE1-mediated regulation of the voltage dependence of activation of the KV7.1 channel.

Activation of Kv7 channels as a novel mechanism for NO/cGMP‐induced pulmonary vasodilation

“…K v 7.5 currents expressed in COS7 were recorded using the perforated‐patch configuration of the patch‐clamp technique (amphotericin B, 125 μg·ml −1 in the internal solution) to prevent rundown using an Axopatch 200B amplifier (Axon Instruments) as previously described (Moreno et al, ; Oliveras et al, ). The intracellular pipette filling solution contained (in mM) K‐aspartate 80, KCl 50, phosphocreatine 3, KH 2 PO 4 10, MgATP 3, HEPES‐K 10, and EGTA 5 and was adjusted to pH 7.25 with KOH.…”

Ventricular voltage‐gated ion channels: Detection, characteristics, mechanisms, and drug safety evaluation

“…147 LQT1, the most common genotype-positive LQTS, is associated with LOF mutations in the KCNQ1encoded Kv7.1 α subunit and is often triggered by β-AR stimulation. 148 Trafficking defects, 149 gating defects, [150][151][152] or permeation disruption 153 have been postulated to be the mechanism of decreasing I Ks or hindering I Ks currents at physiologically relevant membrane potentials but limiting the upregulation of I Ks by PKA activation. 145 Because CaM regulates channel gating by interacting with voltage sensor domains, mutations impair CaM binding (located near the IQ motif of KCNQ1 C-terminus) and alter both channel assembly and gating, thus decreasing I Ks current density and contributing to LQT1.…”