An intracellular hydrophobic nexus critical for hERG1 channel slow deactivation.

Slow deactivation is a critical property of voltage-gated K+ channels encoded by the human Ether-à-go-go-Related Gene 1 (hERG). hERG1 channel deactivation is modulated by interactions between intracellular N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBh) domains. The PAS domain is multipartite, comprising a globular domain (gPAS; residues 26-135) and an N-terminal PAS-cap that is further subdivided into an initial unstructured "tip" (residues 1-12) and an amphipathic α-helical region (residues 13-25). Although the PAS-cap tip has long been considered the effector of slow deactivation, how its position near the gating machinery is controlled has not been elucidated. Here, we show that a triad of hydrophobic interactions among the gPAS, PAS-cap α helix, and the CNBh domains is required to support slow deactivation in hERG1. The primary sequence of this "hydrophobic nexus" is highly conserved among mammalian ERG channels but shows key differences to fast-deactivating Ether-à-go-go 1 (EAG1) channels. Combining sequence analysis, structure-directed mutagenesis, electrophysiology, and molecular dynamics simulations, we demonstrate that polar serine substitutions uncover an intermediate deactivation mode that is also mimicked by deletion of the PAS-cap α helix. Molecular dynamics simulation analyses of the serine-substituted channels show an increase in distance among the residues of the hydrophobic nexus, a rotation of the intracellular gating ring, and a retraction of the PAS-cap tip from its receptor site near the voltage sensor domain and channel gate. These findings provide compelling evidence that the hydrophobic nexus coordinates the respective components of the intracellular gating ring and positions the PAS-cap tip to control hERG1 deactivation gating.

Conformation-sensitive antibody reveals an altered cytosolic PAS/CNBh assembly during hERG channel gating.

The human ERG (hERG) K+ channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS-CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain.

A stable cell line inducibly expressing hERG1a/1b heteromeric channels.

Heterologously expressed hERG channels represent a mainstay of in vitro drug safety screens intended to mitigate risk of cardiac IKr block and sudden cardiac death. This is true even as more channel types are adopted as part of the Comprehensive in vitro Proarrhythmia Assay (CiPA) intended to elevate specificity and thus enhance throughput of promising lead drugs. Until now, hERG1a homomeric channels have been used as a proxy for IKr despite a wealth of evidence showing that hERG1a/1b heteromers better represent native channels in terms of protein abundance and channel biophysical and pharmacological properties. Past efforts to create a stable hERG1a/1b cell line were met with unpredictable silencing of hERG1b expression despite stable integration of the gene into the HEK293 cell genome. Here we report a new cell line stably expressing hERG1a, with hERG1b reliably controlled by an inducible promoter sensitive to doxycycline. Co-immunoprecipitation, Western blot analysis and patch-clamp electrophysiology confirm the heteromeric composition of the expressed channels. Association with hERG1b was found to promote hERG1a protein levels and enhance membrane current levels. Optimal conditions for drug screening and experimental investigation were achieved at 24 h exposure to 100 ng/ml doxycycline. Differences in pharmacological sensitivity between homomeric and heteromeric channels were observed for dofetilide and ebastine, but not fluoxetine, as evaluated by their IC50 values. Using these values in the O'Hara-Rudy-CiPA in silico model revealed discrepancies in pro-arrhythmia risk, implying the hERG1a homomeric platform overestimates risk for these two drugs. Dofetilide block was use-dependent and faster for hERG1a/1b than hERG1a channels, whereas ebastine showed considerable block at rest and had a slower progression for hERG1a/1b channels. The hERG1a/1b cell line thus represents an advanced model for contemporary drug safety screening assays such as CiPA that employ IC50 values to estimate risk of proarrhythmia in computational models of ventricular cardiomyocytes. This novel technology fulfills an unmet need to enhance specificity and foster a safe yet expanded drug development pipeline.