HERG Channel (Dys)function Revealed by Dynamic Action Potential Clamp Technique

Abstract

The human ether-a-go-go-related gene (HERG) encodes the rapid component of the cardiac delayed rectifier potassium current (I(Kr)). Per-Arnt-Sim domain mutations of the HERG channel are linked to type 2 long-QT syndrome. We studied wild-type and/or type 2 long-QT syndrome-associated mutant (R56Q) HERG current (I(HERG)) in HEK-293 cells, at both 23 and 36 degrees C. Conventional voltage-clamp analysis revealed mutation-induced changes in channel kinetics. To assess functional implication(s) of the mutation, we introduce the dynamic action potential clamp technique. In this study, we effectively replace the native I(Kr) of a ventricular cell (either a human model cell or an isolated rabbit myocyte) with I(HERG) generated in a HEK-293 cell that is voltage-clamped by the free-running action potential of the ventricular cell. Action potential characteristics of the ventricular cells were effectively reproduced with wild-type I(HERG), whereas the R56Q mutation caused a frequency-dependent increase of the action potential duration in accordance with the clinical phenotype. The dynamic action potential clamp approach also revealed a frequency-dependent transient wild-type I(HERG) component, which is absent with R56Q channels. This novel electrophysiological technique allows rapid and unambiguous determination of the effects of an ion channel mutation on the ventricular action potential and can serve as a new tool for investigating cardiac channelopathies.

Rescue of Aberrant Gating by a Genetically Encoded PAS (Per-Arnt-Sim) Domain in Several Long QT Syndrome Mutant Human Ether-á-go-go-related Gene Potassium Channels

“…6A, top) showed that hERG R56Q generated a larger outward current than WT hERG channels that peaked earlier during the ramp repolarization phase (Fig. 6, A, C, and D, p Ͻ 0.05), but declined at a faster rate presumably due to its faster deactivation kinetics, similar to what has been previously described (14,24). We next tested whether NPAS rescued changes in resurgent current.…”

“…The LQT2 mutations we selected were based on previous findings demonstrating that when point mutations were made at the selected residues, either to a LQT2-associated mutation or to alanine, the mutant hERG channels exhibited altered deactivation kinetics in Xenopus oocytes (6,(13)(14)(15). We also included K28E, R56Q, and M124R, which were previously investigated in mammalian cells (22)(23)(24). We first measured functional expression of each hERG PAS-LQT2 channel using whole cell patch clamp recordings ( Fig.…”

Concerted All-or-none Subunit Interactions Mediate Slow Deactivation of Human ether-à-go-go-related Gene K+ Channels

“…Thus, R4A/R5A acts in a dominant manner to disrupt the slow kinetics of hERG1 channel deactivation. The LQT2-associated point mutation R56Q, located in the PAS domain of hERG1, was previously reported to increase the rate of channel deactivation when expressed in Xenopus oocytes (8) and to increase the rate of activation and deactivation but not inactivation when channels were heterologously expressed in HEK-293 cells (34). To further explore how a mutation in the PAS domain affects the rate of deactivation, R56Q subunits were placed in either position 1 (R56Q 1 /WT 3 ) or position 3 (WT 2 /R56Q 1 /WT 1 ) of a concatenated tetramer, and the current conducted by these channels was compared with R56Q monomer and R56Q 4 channels.…”

Advances in patch clamp technique: towards higher quality and quantity

“…The analysed cardiac ionic current is specifically inhibited (or abolished if a model of cardiomyocyte is used) and replaced by an ionic current measured in a cell line, e.g. in the human embryonic kidney (HEK) 293 cells in Berecki et al (2005). On the surface of these cells, only the required ionic channel is expressed, either wild-type or mutant.…”

“…The cell line cell is stimulated with AP waveform continuously recorded from the cardiomyocyte (or generated by the model). Berecki et al (2005) used this technique to readily and unambiguously determine the effect of hERG mutation R56Q (identified in a patient with long QT syndrome) on AP configuration in particular layers of ventricular myocardium. Electrophysiological properties of R56Q-hERG mutation found out with the conventional whole-cell patch clamp technique showed controversial changes of AP duration.…”