Identification and characterization of a compound that protects cardiac tissue from human Ether-à-go-go-related gene (hERG)-related drug-induced arrhythmias.

The human Ether-à-go-go-related gene (hERG)-encoded K(+) current, I(Kr) is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I(Kr) block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC(70) of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC(50) of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.

The evolutionarily conserved residue A653 plays a key role in HERG channel closing.

Human ether-a-go-go-related gene (HERG) encodes the rapid, outwardly rectifying K(+) current I(Kr) that is critical for repolarization of the cardiac action potential. Congenital HERG mutations or unintended pharmaceutical block of I(Kr) can lead to life-threatening arrhythmias. Here, we assess the functional role of the alanine at position 653 (HERG-A653) that is highly conserved among evolutionarily divergent K(+) channels. HERG-A653 is close to the 'glycine hinge' implicated in K(+) channel opening, and is flanked by tyrosine 652 and phenylalanine 656, which contribute to the drug binding site. We substituted an array of seven (I, C, S, G, Y, V and T) amino acids at position 653 and expressed individual variants in heterologous systems to assess changes in gating and drug binding. Substitution of A653 resulted in negative shifts of the V(1/2) of activation ranging from -23.6 (A653S) to -62.5 (A653V) compared to -11.2 mV for wild-type (WT). Deactivation was also drastically altered: channels with A653I/C substitutions exhibited delayed deactivation in response to test potentials above the activation threshold, while A653S/G/Y/V/T failed to deactivate under those conditions and required hyperpolarization and prolonged holding potentials at -130 mV. While A653S/G/T/Y variants showed decreased sensitivity to the I(Kr) inhibitor dofetilide, these changes could not be correlated with defects in channel closure. Homology modelling suggests that in the closed state, A653 forms tight contacts with several residues from the neighbouring subunit in the tetramer, playing a key role in S6 helix packing at the narrowest part of the vestibule. Our study suggests that A653 plays an important functional role in the outwardly rectifying gating behaviour of HERG, supporting channel closure at membrane potentials negative to the channel activation threshold.

Functional Interactions between Distinct Sodium Channel Cytoplasmic Domains through the Action of Calmodulin.

Sodium channels are fundamental signaling molecules in excitable cells, and are molecular targets for local anesthetic agents and intracellular free Ca(2+) ([Ca(2+)](i)). Two regions of Na(V)1.5 have been identified previously as [Ca(2+)](i)-sensitive modulators of channel inactivation. These include a C-terminal IQ motif that binds calmodulin (CaM) in different modes depending on Ca(2+) levels, and an immediately adjacent C-terminal EF-hand domain that directly binds Ca(2+). Here we show that a mutation of the IQ domain (A1924T; Brugada Syndrome) that reduces CaM binding stabilizes Na(V)1.5 inactivation, similarly and more extensively than even reducing [Ca(2+)](i). Because the DIII-DIV linker is an essential structure in Na(V)1.5 inactivation, we evaluated this domain for a potential CaM binding interaction. We identified a novel CaM binding site within the linker, validated its interaction with CaM by NMR spectroscopy, and revealed its micromolar affinity by isothermal titration calorimetry. Mutation of three consecutive hydrophobic residues (Phe(1520)-Ile(1521)-Phe(1522)) to alanines in this CaM-binding domain recapitulated the electrophysiology phenotype observed with mutation of the C-terminal IQ domain: Na(V)1.5 inactivation was stabilized; moreover, mutations of either CaM-binding domain abolish the well described stabilization of inactivation by lidocaine. The direct physical interaction of CaM with the C-terminal IQ domain and the DIII-DIV linker, combined with the similarity in phenotypes when CaM-binding sites in either domain are mutated, suggests these cytoplasmic structures could be functionally coupled through the action of CaM. These findings have bearing upon Na(+) channel function in genetically altered channels and under pathophysiologic conditions where [Ca(2+)](i) impacts cardiac conduction.

Genetic screening in C. elegans identifies rho-GTPase activating protein 6 as novel HERG regulator.

The human ether-a-go-go related gene (HERG) constitutes the pore forming subunit of I(Kr), a K(+) current involved in repolarization of the cardiac action potential. While mutations in HERG predispose patients to cardiac arrhythmias (Long QT syndrome; LQTS), altered function of HERG regulators are undoubtedly LQTS risk factors. We have combined RNA interference with behavioral screening in Caenorhabditis elegans to detect genes that influence function of the HERG homolog, UNC-103. One such gene encodes the worm ortholog of the rho-GTPase activating protein 6 (ARHGAP6). In addition to its GAP function, ARHGAP6 induces cytoskeletal rearrangements and activates phospholipase C (PLC). Here we show that I(Kr) recorded in cells co-expressing HERG and ARHGAP6 was decreased by 43% compared to HERG alone. Biochemical measurements of cell-surface associated HERG revealed that ARHGAP6 reduced membrane expression of HERG by 35%, which correlates well with the reduction in current. In an atrial myocyte cell line, suppression of endogenous ARHGAP6 by virally transduced shRNA led to a 53% enhancement of I(Kr). ARHGAP6 effects were maintained when we introduced a dominant negative rho-GTPase, or ARHGAP6 devoid of rhoGAP function, indicating ARHGAP6 regulation of HERG is independent of rho activation. However, ARHGAP6 lost effectiveness when PLC was inhibited. We further determined that ARHGAP6 effects are mediated by a consensus SH3 binding domain within the C-terminus of HERG, although stable ARHGAP6-HERG complexes were not observed. These data link a rhoGAP-activated PLC pathway to HERG membrane expression and implicate this family of proteins as candidate genes in disorders involving HERG.

In vivo identification of genes that modify ether-a-go-go-related gene activity in Caenorhabditis elegans may also affect human cardiac arrhythmia.

Human ether-a-go-go-related gene (HERG) encodes the pore-forming subunit of I(Kr), a cardiac K(+) channel. Although many commonly used drugs block I(Kr), in certain individuals, this action evokes a paradoxical life-threatening cardiac rhythm disturbance, known as the acquired long QT syndrome (aLQTS). Although aLQTS has become the leading cause of drug withdrawal by the U.S. Food and Drug Administration, DNA sequencing in aLQTS patients has revealed HERG mutations only in rare cases, suggesting that unknown HERG modulators are often responsible. By using the worm Caenorhabditis elegans, we have developed in vivo behavioral assays that identify candidate modulators of unc-103, the worm HERG orthologue. By using RNA-interference methods, we have shown that worm homologues of two HERG-interacting proteins, Hyperkinetic and K channel regulator 1 (KCR1), modify unc-103 function. Examination of the human KCR1 sequence in patients with drug-induced cardiac repolarization defects revealed a sequence variation (the substitution of isoleucine 447 by valine, I447V) that occurs at a reduced frequency (1.1%) relative to a matched control population (7.0%), suggesting that I447V may be an allele for reduced aLQTS susceptibility. This clinical result is supported by in vitro studies of HERG dofetilide sensitivity by using coexpression of HERG with wild-type and I447V KCR1 cDNAs. Our studies demonstrate the feasibility of using C. elegans to assay and potentially identify aLQTS candidate genes.

Functional interaction between extracellular sodium, potassium and inactivation gating in HERG channels.

We have studied the interaction between extracellular K(+) (K(+)(o)) and extracellular Na(+) (Na(+)(o)) in human ether-à-go-go related gene (HERG)-encoded K(+) channels expressed in Chinese hamster ovary (CHO-K1) cells, using the whole-cell voltage clamp technique. Prior studies indicate that Na(+)(o) potently inhibits HERG current (IC(50) 3 mm) by binding to an outer pore site, and also speeds recovery from inactivation. In this study, we sought to explore the relationship between the Na(+)(o) effect on recovery and Na(+)(o) inhibition of HERG current, and to determine whether inactivation gating plays a critical role in Na(+)(o) inhibition of HERG current. Na(+)(o) concentration-response relationships for current inhibition and speeding of recovery were different, with Na(+)(o) less potent at speeding recovery. Na(+)(o) inhibition of HERG current was relieved by physiological [K(+)](o), while Na(+)(o) speeded recovery from inactivation similarly in the absence or presence of physiological [K(+)](o). To examine the link between Na(+)(o) block and inactivation using an independent approach, we studied hyperpolarization-activated currents uncoupled from inactivation in the S4-S5 linker mutant D540K. Depolarization-activated D540K currents were inhibited by Na(+)(o), while hyperpolarization-activated currents were augmented by Na(+)(o). This result reveals a direct link between Na(+)(o) inhibition and a depolarization-induced conformational change, most likely inactivation. We attempted to simulate the disparate concentration-response relationships for the two effects of Na(+)(o) using a kinetic model that included Na(+)(o) site(s) affected by permeation and gating. While a model with only a single dynamic Na(+)(o) site was inadequate, a model with two distinct Na(+)(o) sites was sufficient to reproduce the data.

Extracellular sodium interacts with the HERG channel at an outer pore site.

Most voltage-gated K(+) currents are relatively insensitive to extracellular Na(+) (Na(+)(o)), but Na(+)(o) potently inhibits outward human ether-a-go-go-related gene (HERG)-encoded K(+) channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429-30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na(+) (Na(+)(i)) and extracellular Ba(2+) (Ba(2+)(o)), to define a site where Na(+)(o) interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na(+)(o) was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K(+) selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na(+)(o). Quantitatively similar effects of Na(+)(i) to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na(+)(o) sensitivity, this result suggested that Na(+)(o) and Na(+)(i) act at different sites. Extracellular Ba(2+) (Ba(2+)(o)) blocks K(+) channel pores, and thereby serves as a useful probe of K(+) channel structure. HERG channel inactivation promotes relief of Ba(2+) block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265-278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na(+)(o) action. A remote allosteric model predicts that Na(+)(o) will speed relief of Ba(2+)(o) block by promoting inactivation. Instead, Na(+)(o) slowed Ba(2+) egress and Ba(2+) relieved Na(+)(o) inhibition, consistent with Na(+)(o) binding to an outer pore site. The apparent affinities of the outer pore for Na(+)(o) and K(+)(o) as measured by slowing of Ba(2+) egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na(+)(o) inhibition. Na(+)(o) inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A.