Abstract
Human Ether á go-go Related Gene potassium channels form the rapid component of the delayed-rectifier (IKr) current in the heart. The N-terminal ‘eag’ domain, which is composed of a Per-Arnt-Sim (PAS) domain and a short PAS-cap region, is a critical regulator of hERG channel function. In previous studies, we showed that isolated eag (i-eag) domains rescued the dysfunction of long QT type-2 associated mutant hERG R56Q channels, by substituting for defective eag domains, when the channels were expressed in Xenopus oocytes or HEK 293 cells.Here, our goal was to determine whether the rescue of hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes. We expressed hERG R56Q channels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and measured electrical properties of the cells with whole-cell patch-clamp recordings. We found that, like in non-myocyte cells, hERG R56Q had defective, fast closing (deactivation) kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics of hERG R56Q channels in hiPSC-CMs. hERG R56Q channels prolonged the AP of hiPSCs, and the AP was shortened by co-expression of i-eag domains and hERG R56Q channels. We measured robust Förster Resonance Energy Transfer (FRET) between i-eag domains tagged with Cyan fluorescent protein (CFP) and hERG R56Q channels tagged with Citrine fluorescent proteins (Citrine), indicating their close proximity at the cell membrane in live iPSC-CMs. Together, functional regulation and FRET spectroscopy measurements indicated that i-eag domains interacted directly with hERG R56Q channels in hiPSC-CMs. These results mean that the regulatory role of i-eag domains is conserved in the cellular environment of human cardiomyocytes, indicating that i-eag domains may be useful as a biological therapeutic.
Highlights
Human ether à go-go Related Gene K+ channels are members of the voltage-activated (Kv) family of channels, which are characterized by opening in response to membranePLOS ONE | DOI:10.1371/journal.pone.0123951 April 29, 2015Eag Domains Regulate LQT Mutant hERG Channels depolarization and high selectivity for K+ ions
We analyzed the tail current produced by a step to 60 mV from a potential of -80 mV and the decay was fit with a double exponential function to derive fast and slow time constants of deactivation. hERG R56Q currents had a faster time course of decay compared with that of wild-type hERG (WT hERG) at -50 mV and deactivation time course was partially restored by i-eag domains (Fig 1B, 1C and 1D; Table 2)
We report here that hERG R56Q channels heterologously expressed in hiPSC-CMs had faster deactivation kinetics and a less-rectifying I-V relationship and a larger and earlier peak current in response to an action potential (AP) waveform than that of WT hERG channels, similar to the properties of hERG R56Q expressed in Xenopus oocytes or HEK293 cells
Summary
Eag Domains Regulate LQT Mutant hERG Channels depolarization and high selectivity for K+ ions. Like other Kv channels, hERG channels have six transmembrane domains and intracellular N- and C-terminal regions[1]. The CNBHD is structurally similar to that of the CNG and HCN channels, but unlike those channels, the hERG CNBHD only weakly associates with cyclic nucleotides and does not undergo regulation by cyclic nucleotides[2]. In place of a cyclic nucleotide, the CNBHD in the hERG family of channels is bound by an intrinsic ligand, which is primarily composed of a three amino acid piece of the CNBHD itself[3,4,5,6]
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