hERG 1b is critical for human cardiac repolarization

Abstract

The human ether-à-go-go-related gene (hERG; or KCNH2) encodes the voltage-gated potassium channel underlying I Kr , a repolarizing current in the heart. Mutations in KCNH2 or pharmacological agents that reduce I Kr slow action potential (AP) repolarization and can trigger cardiac arrhythmias associated with long QT syndrome. Two channel-forming subunits encoded by KCNH2 (hERG 1a and 1b) are expressed in cardiac tissue. In heterologous expression systems, these subunits avidly coassemble and exhibit biophysical and pharmacological properties distinct from those of homomeric hERG 1a channels. Despite these findings, adoption of hERG 1a/1b heteromeric channels as a model for cardiac I Kr has been hampered by the lack of evidence for a direct functional role for the 1b subunit in native tissue. In this study, we measured I Kr and APs at physiological temperature in cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs). We found that specific knockdown of the 1b subunit using shRNA caused reductions in 1b mRNA, 1b protein levels, and I Kr magnitude by roughly one-half. AP duration was increased and AP variability was enhanced relative to controls. Early afterdepolarizations, considered cellular substrates for arrhythmia, were also observed in cells with reduced 1b expression. Similar behavior was elicited when channels were effectively converted from heteromers to 1a homomers by expressing a fragment corresponding to the 1a-specific N-terminal Per-Arnt-Sim domain, which is omitted from hERG 1b by alternate transcription. These findings establish that hERG 1b is critical for normal repolarization and that loss of 1b is proarrhythmic in human cardiac cells.T he human ether-à-go-go-related gene (hERG; or KCNH2) encodes the voltage-gated potassium channel underlying a native cardiac current known as I Kr (1, 2). Mutations in the gene or drugs that block the channel can diminish I Kr and slow ventricular repolarization, thus prolonging the QT interval on the surface electrocardiogram and causing long QT syndrome (LQTS) (1-3). Individuals with LQTS have an increased risk for torsades de pointes arrhythmia, syncope, and sudden cardiac death. Since 2005, a cell-based assay expressing the hERG 1a isoform as a proxy for I Kr has been the cornerstone of drug safety screening for new drug development (4). Thus, whether current drug safety assays accurately represent the native channel is of paramount importance.The first studies of heterologously expressed hERG channels described biophysical (1, 2) and pharmacological (2, 5) properties uniquely characteristic of cardiac I Kr . Subsequently, alternate transcripts of KCNH2 in mouse and human heart were shown to encode two subunits: 1a (the original isolate) and 1b (6, 7). In the hERG 1b transcript, an alternate 5′ exon replaces 1a exons 1-5, resulting in a shorter, unique N terminus that lacks a Per-Arnt-Sim (PAS) domain (also known as the ether-à-go-go domain) (8, 9). In heterologous systems, hERG 1b subunits avidly associate with hERG 1a but fail as homomer...

Cotranslational association of mRNA encoding subunits of heteromeric ion channels

“…In isolated cardiomyocytes, I Kr channels can be functionally converted to homomeric 1a-like channels by exogenous expression of the 1a-specific Per-Arnt-Sim (PAS) domain. The result is diminished repolarizing current amplitude and cellular manifestations of proarrhythmia, including prolonged action potential duration (APD), APD variability, and early afterdepolarizations (9). These findings reinforce the importance of both hERG 1a and 1b subunits in cardiac repolarization.…”

“…These experiments independently confirm that the mRNA transcripts targeted by the shRNA in the iPSC-CMs were actively undergoing protein translation. The observation that either shRNA can reduce current magnitude underscores the importance of both subunits in I Kr channel function (9).…”

“…Surprisingly, the 1b-specific shRNA reduced both 1a and 1b transcripts when they were coexpressed in HEK293 cells (9), suggesting the transcripts were associated. Here, we tested this hypothesis and the more general idea that heteromeric channel assembly is mediated by a complex comprising the encoding Significance Many ion channels are composed of related but structurally distinct membrane protein subunits, each determining the functional properties of the channel.…”

“…Cardiac I Kr , a repolarizing current in the heart (4), is produced by heteromeric assemblies of human ether-à-go-go-related gene (hERG) 1a and 1b subunits (5)(6)(7)(8)(9)(10). Perturbations of I Kr by congenital mutations in the KCNH2/hERG1 gene or by drug block can prolong the ventricular action potential and associated QT interval to cause long QT syndrome and sudden cardiac death (11)(12)(13).…”

“…To determine whether the hERG 1a and 1b transcripts similarly associate under native conditions, we used iPSC-CMs in which native I Kr is produced by heteromeric channels comprising hERG 1a and 1b subunits (9). In the iPSC-CMs, like HEK293 cells, either transcript-specific Both subunits were reduced, corresponding to the decrease of mRNA in B (n = 3-5).…”