Dominant negative consequences of a hERG 1b-specific mutation associated with intrauterine fetal death

Abstract

The human ether-a-go-go related gene (hERG) encodes two subunits, hERG 1a and hERG 1b, that combine in vivo to conduct the rapid delayed rectifier potassium current (IKr). Reduced IKr slows cardiac action potential (AP) repolarization and is an underlying cause of cardiac arrhythmias associated with long QT syndrome (LQTS). Although the physiological importance of hERG 1b has been elucidated, the effects of hERG 1b disease mutations on cardiac IKr and AP behavior have not been described. To explore the disease mechanism of a 1b-specific mutation associated with a case of intrauterine fetal death, we examined the effects of the 1b-R25W mutation on total protein, trafficking and membrane current levels in HEK293 cells at physiological temperatures. By all measures the 1b-R25W mutation conferred diminished expression, and exerted a temperature-sensitive, dominant-negative effect over the WT hERG 1a protein with which it was co-expressed. Membrane currents were reduced by 60% with no apparent effect on voltage dependence or deactivation. The dominant-negative effects of R25W were demonstrated in iPSC-CMs, where 1b-R25W transfection diminished native IKr compared to controls. R25W also slowed AP repolarization, and increased AP triangulation and variability in iPSC-CMs, reflecting cellular manifestations of pro-arrhythmia. These data demonstrate that R25W is a dominant-negative mutation with significant pathophysiological consequences, and provide the first direct link between hERG 1b mutation and cardiomyocyte dysfunction.

Mitragynine, an euphoric compound inhibits hERG1a/1b channel current and upregulates the complexation of hERG1a-Hsp90 in HEK293-hERG1a/1b cells

“…In contrast, fluoxetine appeared to be a more potent blocker of hERG1a/1b than hERG1a channels 33 . Furthermore, multiple studies have confirmed the importance of hERG1a/1b in maintaining normal physiological function in the human heart 31,32,34,51 . Hence, apart from hERG1a channel, it is important to investigate inhibition potential of drugs on hERG1a/1b channels given the different gating properties and drug sensitivities of the isoforms.…”

Localization and functional consequences of a direct interaction between TRIOBP-1 and hERG proteins in the heart

“…To determine whether native I Kr is affected by TRIOBP-1 overexpression, we performed patch-clamp electrophysiology experiments in cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) (Harley et al, 2016;Jones et al, 2014Jones et al, , 2016Ma et al, 2011). I Kr , measured as an E-4031sensitive current (see Materials and Methods), was markedly reduced by TRIOBP-1 overexpression (Fig.…”

“…iPSC-CM recordings were conducted at 5-30 days post plating. All iPSC-CM recordings were completed 48-96 h post transfection at 36±1°C using whole-cell patch-clamp as described previously (Jones et al, 2016). We only recorded from cells that displayed GFP fluorescence, which corresponds to successful Kir2.1 transduction.…”

hERG1a and hERG1b potassium channel subunits directly interact and preferentially form heteromeric channels

“…Interactions between hERG1a and hERG1b isoforms may have relevance for drug block and acquired LQTS because there is a difference in affinity for drugs between currents from HEK293 cells expressing hERG1a compared with hERG1a plus hERG1b (32). Furthermore, hERG1a and hERG1b assembly may differently affect isoform-specific LQT mutations that selectively disrupt hERG1a (33,34) or hERG1b (15,35).…”