Serine mutation of a conserved threonine in the hERG K+ channel S6-pore region leads to loss-of-function through trafficking impairment

Ehab Al-Moubarak,Yihong Zhang,Christopher E Dempsey,Henggui Zhang,Stephen C Harmer,Jules C Hancox

doi:10.1016/j.bbrc.2020.04.003

Abstract

The human Ether-à-go-go Related Gene (hERG) encodes a potassium channel responsible for the cardiac rapid delayed rectifier K+ current, IKr, which regulates ventricular repolarization. Loss-of-function hERG mutations underpin the LQT2 form of congenital long QT syndrome. This study was undertaken to elucidate the functional consequences of a variant of uncertain significance, T634S, located at a highly conserved position at the top of the S6 helix of the hERG channel. Whole-cell patch-clamp recordings were made at 37 °C of hERG current (IhERG) from HEK 293 cells expressing wild-type (WT) hERG, WT+T634S and hERG-T634S alone. When the T634S mutation was expressed alone little or no IhERG could be recorded. Co-expressing WT and hERG-T634S suppressed IhERG tails by ∼57% compared to WT alone, without significant alteration of voltage dependent activation of IhERG. A similar suppression of IhERG was observed under action potential voltage clamp. Comparable reduction of IKr in a ventricular AP model delayed repolarization and led to action potential prolongation. A LI-COR® based On/In-Cell Western assay showed that cell surface expression of hERG channels in HEK 293 cells was markedly reduced by the T634S mutation, whilst total cellular hERG expression was unaffected, demonstrating impaired trafficking of the hERG-T634S mutant. Incubation with E−4031, but not lumacaftor, rescued defective hERG-T634S channel trafficking and IhERG density. In conclusion, these data identify hERG-T634S as a rescuable trafficking defective mutation that reduces IKr sufficiently to delay repolarization and, thereby, potentially produce a LQT2 phenotype.

Highlights

Electrical repolarization determines the duration of ventricular action potentials and, thereby, the length of the QT interval on the electrocardiogram
Most human Ethera-go-go Related Gene (hERG) mutations linked to congenital LQT2 are missense mutations, the majority of which impair channel transport within the cell; misfolded hERG proteins become retained within the endoplasmic reticulum, thereby limiting the number of functional channels in the cell membrane [4,6]
Mutations to nearby residues in hERG have been associated with LQTS [27] and the T634I and T634A mutations at the same position in the hERG protein have previously been associated with LQT2 [6,12]

Summary

Introduction

Electrical repolarization determines the duration of ventricular action potentials and, thereby, the length of the QT interval on the electrocardiogram. Of the potassium channel currents that contribute to ventricular repolarization, the rapid delayed rectifier current, IKr, appears to be notable. The human Ethera-go-go Related Gene (hERG; alternative nomenclature KCNH2) encodes channels that mediate IKr [1,2]. Loss-of-function hERG mutations lead to the LQT2 form of congenital long QT syndrome [3,4], whilst gain-of-function hERG mutations underpin the SQT1 form of short QT syndrome [5]. Most hERG mutations linked to congenital LQT2 are missense mutations, the majority of which impair channel transport within the cell (trafficking); misfolded hERG proteins become retained within the endoplasmic reticulum, thereby limiting the number of functional channels in the cell membrane [4,6]. Over 1000 hERG variants exist on publicly available databases such as ClinVar, but functional data are available for only a fraction of these

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Conclusion