T75M-KCNJ2 mutation causing Andersen–Tawil syndrome enhances inward rectification by changing Mg²⁺ sensitivity

Abstract 

Andersen–Tawil syndrome (ATS) is a multisystem inherited disease exhibiting periodic paralysis, cardiac arrhythmias, and dysmorphic features. In this study, we characterized the KCNJ2 channels with an ATS mutation (T75M) which is associated with cardiac phenotypes of bi-directional ventricular tachycardia, syncope, and QT_c prolongation. Confocal imaging of GFP-KCNJ2 fusion proteins showed that the T75M mutation impaired membrane localization of the channel protein, which was restored by co-expression of WT channels with T75M channels. Whole-cell patch-clamp experiments in CHO-K1 cells showed that the T75M mutation produced a loss-of-function of the channel. When both WT and the T75M were co-expressed, the T75M mutation showed dominant-negative effects on inward rectifier K⁺ current densities, with prominent suppression of outward currents at potentials between 0 mV and +80 mV over the E_K. Inside-out patch experiments in HEK293T cells revealed that co-expression of WT and the T75M channels enhanced voltage-dependent block of the channels by internal Mg²⁺, resulting in enhanced inward rectification at potentials 50 mV more positive than the E_K. We suggest that the T75M mutation causes dominant-negative suppression of the co-expressed WT KCNJ2 channels. In addition, the T75M mutation caused alteration of gating kinetics of the mutated KCNJ2 channels, i.e., increased sensitivity to intracellular Mg²⁺ and resultant enhancement of inward rectification. The data presented suggest that the mutation may influence clinical features, but it does not directly show this.

Abbreviations: ATS, Andersen–Tawil syndrome, GFP, green fluorescent protein, WT, wild type, CHO-K1 cell, Chinese hamster ovary-K1 cell, HEK293T cell, human embryonic kidney 293T cells, E_K, potassium reversal potential, I_K1, inward rectifying potassium current, PIP₂, phosphatidylinositol 4,5-diphosphate, ECG, electrocardiogram, PCR, polymerase chain reaction, cDNA, complementary deoxyribonucleic acid, PBS, phosphate buffered saline, HEPES, 2-[4-(2-hydroxyethyl)-1-piperadinyl] ethansulfonic acid, ATP, adenosine triphosphate, I–V, current to voltage, NMDG-Cl, N-methyl-d-glucamine chloride, EDTA, ethylenediaminetetracetic acid, S.E.M., standard error of mean, ANOVA, analysis of variance, Thr, threonine, Met, methionine, C, cytosine, T, thymine, Glu, glutamic acid, Asp, aspartic acid, Asn, asparagine, LQT, long QT syndrome, SQT, short QT syndrome

Keywords: Andersen–Tawil syndrome, QT_c prolongation, KCNJ2, The Kir2.1 (I_K1) channel, Inward rectification, Magnesium, Spermine