Molecular determinants for activation of human ether-a-go-go-related gene 1 potassium channels by 3-nitro-n-(4-phenoxyphenyl) benzamide.
ABSTRACT: Human ether-à-go-go-related gene 1 (hERG1) channels mediate repolarization of cardiac action potentials. Inherited long QT syndrome (LQTS) caused by loss-of-function mutations, or unintended blockade of hERG1 channels by many drugs, can lead to severe arrhythmia and sudden death. Drugs that activate hERG1 are a novel pharmacological approach to treat LQTS. 3-Nitro-n-(4-phenoxyphenyl) benzamide [ICA-105574 (ICA)] has been discovered to activate hERG1 by strong attenuation of pore-type inactivation. Here, we used scanning mutagenesis of hERG1 to identify the molecular determinants of ICA action. Three mutations abolished the activator effects of 30 ?M ICA, including L622C in the pore helix, F557L in the S5 segment, and Y652A in the S6 segment. One mutation in S6 (A653M) switched the activity of ICA from an activator to an inhibitor, revealing its partial agonist activity. This was confirmed by showing that the noninactivating mutant hERG1 channel (G628C/S631C) was inhibited by ICA and that the addition of the F557L mutation rendered the channel drug-insensitive. Simulated molecular docking of ICA to homology models of hERG1 corroborated the scanning mutagenesis findings. Together, our findings indicate that ICA is a mixed agonist of hERG1 channels. Activation or inhibition of currents is mediated by the same or overlapping binding site located in the pore module between two adjacent subunits of the homotetrameric channel.
Project description:During the repolarization phase of a cardiac action potential, hERG1 K(+) channels rapidly recover from an inactivated state then slowly deactivate to a closed state. The resulting resurgence of outward current terminates the plateau phase and is thus a key regulator of action potential duration of cardiomyocytes. The intracellular N-terminal domain of the hERG1 subunit is required for slow deactivation of the channel as its removal accelerates deactivation 10-fold. Here we investigate the stoichiometry of hERG1 channel deactivation by characterizing the kinetic properties of concatenated tetramers containing a variable number of wild-type and mutant subunits. Three mutations known to accelerate deactivation were investigated, including R56Q and R4A/R5A in the N terminus and F656I in the S6 transmembrane segment. In all cases, a single mutant subunit induced the same rapid deactivation of a concatenated channel as that observed for homotetrameric mutant channels. We conclude that slow deactivation gating of hERG1 channels involves a concerted, fully cooperative interaction between all four wild-type channel subunits.
Project description:Activation of human ether-a-go-go-related gene 1 (hERG1) K(+) channels mediates cardiac action potential repolarization. Drugs that activate hERG1 channels represent a mechanism-based approach for the treatment of long QT syndrome, a disorder of cardiac repolarization associated with ventricular arrhythmia and sudden death. Here, we characterize the mechanisms of action and the molecular determinants for binding of RPR260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid] (RPR), a recently discovered hERG1 channel activator. Channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured by using the two-microelectrode voltage-clamp technique. RPR induced a concentration-dependent slowing in the rate of channel deactivation and enhanced current magnitude by shifting the voltage dependence of inactivation to more positive potentials. This mechanism was confirmed by demonstrating that RPR slowed the rate of deactivation, but did not increase current magnitude of inactivation-deficient mutant channels. The effects of RPR on hERG1 kinetics and magnitude could be simulated by reducing three rate constants in a Markov model of channel gating. Point mutations of specific residues located in the S4-S5 linker or cytoplasmic ends of the S5 and S6 domains greatly attenuated or ablated the effects of 3 microM RPR on deactivation (five residues), inactivation (one residue), or both gating mechanisms (four residues). These findings define a putative binding site for RPR and confirm the importance of an interaction between the S4-S5 linker and the S6 domain in electromechanical coupling of voltage-gated K(+) channels.
Project description:The hERG1 gene (Kv11.1) encodes a voltage-gated potassium channel. Mutations in this gene lead to one form of the Long QT Syndrome (LQTS) in humans. Promiscuous binding of drugs to hERG1 is known to alter the structure/function of the channel leading to an acquired form of the LQTS. Expectably, creation and validation of reliable 3D model of the channel have been a key target in molecular cardiology and pharmacology for the last decade. Although many models were built, they all were limited to pore domain. In this work, a full model of the hERG1 channel is developed which includes all transmembrane segments. We tested a template-driven de-novo design with ROSETTA-membrane modeling using side-chain placements optimized by subsequent molecular dynamics (MD) simulations. Although backbone templates for the homology modeled parts of the pore and voltage sensors were based on the available structures of KvAP, Kv1.2 and Kv1.2-Kv2.1 chimera channels, the missing parts are modeled de-novo. The impact of several alignments on the structure of the S4 helix in the voltage-sensing domain was also tested. Herein, final models are evaluated for consistency to the reported structural elements discovered mainly on the basis of mutagenesis and electrophysiology. These structural elements include salt bridges and close contacts in the voltage-sensor domain; and the topology of the extracellular S5-pore linker compared with that established by toxin foot-printing and nuclear magnetic resonance studies. Implications of the refined hERG1 model to binding of blockers and channels activators (potent new ligands for channel activations) are discussed.
Project description:Voltage-gated K(+) channels are tetramers formed by coassembly of four identical or highly related subunits. All four subunits contribute to formation of the selectivity filter, the narrowest region of the channel pore which determines K(+) selective conductance. In some K(+) channels, the selectivity filter can undergo a conformational change to reduce K(+) flux by a mechanism called C-type inactivation. In human ether-a-go-go-related gene 1 (hERG1) K(+) channels, C-type inactivation is allosterically inhibited by ICA-105574, a substituted benzamide. PD-118057, a 2-(phenylamino) benzoic acid, alters selectivity filter gating to enhance open probability of channels. Both compounds bind to a hydrophobic pocket located between adjacent hERG1 subunits. Accordingly, a homotetrameric channel contains four identical activator binding sites. Here we determine the number of binding sites required for maximal drug effect and determine the role of subunit interactions in the modulation of hERG1 gating by these compounds. Concatenated tetramers were constructed to contain a variable number (zero to four) of wild-type and mutant hERG1 subunits, either L646E to inhibit PD-118057 binding or F557L to inhibit ICA-105574 binding. Enhancement of hERG1 channel current magnitude by PD-118057 and attenuated inactivation by ICA-105574 were mediated by cooperative subunit interactions. Maximal effects of the both compounds required the presence of all four binding sites. Understanding how hERG1 agonists allosterically modify channel gating may facilitate mechanism-based drug design of novel agents for treatment of long QT syndrome.
Project description:Activators of human ether-a-go-go-related gene 1 (hERG1) channels, such as (3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid (RPR260243), reverse the effect of hERG1 blockers and shorten the duration of cardiac action potentials. RPR260243 (RPR) slows the rate of deactivation and shifts the voltage dependence of channel inactivation to more positive potentials. We recently mapped the binding site for RPR to several residues located near the cytoplasmic ends of the S5 and S6 helices of the hERG1 subunit. These residues are conserved in the highly homologous ether-a-go-go-related gene 3 (ERG3) subunit; however, RPR blocks ERG3 channels. Here, we compare hERG1 and rat ERG3 (rERG3) channels to explore the molecular basis for differential channel sensitivity to RPR. Channels were heterologously expressed in Xenopus laevis oocytes, and currents were recorded using the two-electrode voltage-clamp technique. Site-directed mutagenesis was used to swap the two residues within the putative binding domain that differed between hERG1 and rERG3. The differential sensitivity of hERG1 and rERG3 channels to the agonist effect of RPR could be accounted for by a single S5 residue (Thr556 in hERG1, Ile558 in rERG3). A Thr in this position favors agonist activity, whereas an Ile reveals a secondary blocking effect of RPR.
Project description:Emerging evidence suggests that K(+) channel inactivation involves coupling between residues in adjacent regions of the channel. Human ether-a-go-go-related gene-1 (hERG1) K(+) channels undergo a fast inactivation gating process that is crucial for maintaining electrical stability in the heart. The molecular mechanisms that drive inactivation in hERG1 channels are unknown. Using alanine scanning mutagenesis, we show that a pore helix residue (Thr-618) that points toward the S5 segment is critical for normal inactivation gating. Amino acid substitutions at position 618 modulate the free energy of inactivation gating, causing enhanced or reduced inactivation. Mutation of an S5 residue that is predicted to be adjacent to Thr-618 (W568L) abolishes inactivation and alters ion selectivity. The introduction of the Thr-618-equivalent residue in Kv1.5 enhances inactivation. Molecular dynamic simulations of the Kv1.2 tetramer reveal van der Waals coupling between hERG1 618- and 568-equivalent residues and a significant increase in interaction energies when threonine is introduced at the 618-equivalent position. We propose that coupling between the S5 segment and pore helix may participate in the inactivation process in hERG1 channels.
Project description:Human ether-a-go-go-related gene 1 (hERG1) K(+) channels mediate repolarization of cardiac action potentials. Unintended block of hERG1 channels by some drugs can prolong the QT interval and induce arrhythmia. Recently, hERG1 channel agonists were discovered and, based on their mechanisms of action can be classified into two types. RPR260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5 trifluorophenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid], a type 1 agonist, binds to residues located near the intracellular end of S5 and S6 transmembrane segments and activates hERG1 channels by a dual mechanism of slowed deactivation and attenuated P-type inactivation. As defined here, type 2 agonists such as PD-118057 [2-(4-[2-(3,4-dichloro-phenyl)-ethyl]-phenylamino)-benzoic acid] attenuate inactivation but do not slow deactivation. At 10 muM, PD-118057 shifted the half-point for inactivation of wild-type hERG1 channels by +19 mV and increased peak outward current by 136%. Scanning mutagenesis and functional characterization of 44 mutant channels expressed in Xenopus oocytes was used to identify the major structural determinants of the binding site for PD-118057. Single mutations of residues in the pore helix (F619) or the S6 segment (L646) of hERG1 eliminated agonist activity. Mutation of a nearby residues in the S6 segment (C643, M645) enhanced drug activity, presumably by reducing steric hindrance for drug binding. Molecular modeling indicates that PD-118057 binds to a hydrophobic pocket formed by L646 of one hERG1 subunit and F619 of an adjacent subunit. We conclude that direct interaction of PD-118057 with the pore helix attenuates fast P-type inactivation and increases open probability of hERG1 channels.
Project description:N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7) is a well-known calmodulin inhibitor used to study calmodulin regulation of intracellular Ca(2+) signalling-related process. Here, we have determined whether W-7 would inhibit human ether gene (hERG or K(v) 11.1) potassium channels, hK(v) 1.5 channels or hK(IR) 2.1 channels expressed in human embryonic kidney (HEK) 293 cells.The hERG channel current, hK(v) 1.5 channel current or hK(IR) 2.1 channel current was recorded with a whole-cell patch clamp technique.It was found that the calmodulin inhibitor W-7 blocked hERG, hK(v) 1.5 and hK(IR) 2.1 channels. W-7 decreased the hERG current (I(hERG) ) in a concentration-dependent manner (IC(50) : 3.5 µM), and the inhibition was more significant at depolarization potentials between +10 and +60 mV. The hERG mutations in the S6 region Y652A and F656V, and in the pore helix S631A, had the IC(50) s of 5.5, 9.8 and 25.4 µM respectively. In addition, the compound inhibited hK(v) 1.5 and hK(IR) 2.1 channels with IC(50) s of 6.5 and 13.4 µM respectively.These results indicate that the calmodulin inhibitor W-7 exerts a direct channel-blocking effect on hERG, hK(v) 1.5 and hK(IR) 2.1 channels stably expressed in HEK 293 cells. Caution should be taken in the interpretation of calmodulin regulation of ion channels with W-7.
Project description:Human ether-à-go-go-related gene 1 (hERG1) channels conduct the rapid delayed rectifier K+ current, I(Kr), an important determinant of action potential repolarization in mammals, including humans. Reduced I(Kr) function caused by mutations in KCNH2 or drug block of hERG1 channels prolongs the QT interval of the electrocardiogram and increases the risk of ventricular fibrillation and sudden cardiac death. Several activators of hERG1 channels have been discovered in recent years. These compounds shorten the duration of cardiac action potentials and have been proposed as a new therapeutic approach for the treatment of acquired or congenital long QT syndrome. We defined previously the mechanism of action of 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643), a compound that increases hERG1 currents by shifting the voltage-dependence of inactivation to more positive potentials. Here, we use scanning mutagenesis of hERG1 and functional characterization of 56 mutant channels heterologously expressed in Xenopus laevis oocytes to define the molecular determinants of the binding site for NS1643. Most point mutations did not alter response to the drug; however, 10 mutant channels had reduced sensitivity, and F619A and I567A exhibited enhanced activation by the drug. Some of these residues form a cluster and, together with molecular modeling, suggest that NS1643 binds to a pocket near the extracellular ends of the S5/S6 segments of two adjacent hERG1 channel subunits. This putative binding site differs from the sites described previously for two other hERG1 activators, (3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid (RPR260243) and 2-(4-[2-(3,4-dichloro-phenyl)-ethyl]-phenylamino)-benzoic acid (PD-118057).
Project description:Human ether-à-go-go-related gene (Kv11.1, or hERG) is a potassium channel that conducts the delayed rectifier potassium current (IKr) during the repolarization phase of cardiac action potentials. hERG channels have a larger pore than other K+channels and can trap many unintended drugs, often resulting in acquired LQTS (aLQTS). R-roscovitine is a cyclin-dependent kinase (CDK) inhibitor that induces apoptosis in colorectal, breast, prostate, multiple myeloma, other cancer cell lines, and tumor xenografts, in micromolar concentrations. It is well tolerated in phase II clinical trials. R-roscovitine inhibits open hERG channels but does not become trapped in the pore. Two-electrode voltage clamp recordings from Xenopus oocytes expressing wild-type (WT) or hERG pore mutant channels (T623A, S624A, Y652A, F656A) demonstrated that compared to WT hERG, T623A, Y652A, and F656A inhibition by 200 ?M R-roscovitine was ~ 48%, 29%, and 73% weaker, respectively. In contrast, S624A hERG was inhibited more potently than WT hERG, with a ~ 34% stronger inhibition. These findings were further supported by the IC50 values, which were increased for T623A, Y652A and F656A (by ~5.5, 2.75, and 42 fold respectively) and reduced 1.3 fold for the S624A mutant. Our data suggest that while T623, Y652, and F656 are critical for R-roscovitine-mediated inhibition, S624 may not be. Docking studies further support our findings. Thus, R-roscovitine's relatively unique features, coupled with its tolerance in clinical trials, could guide future drug screens.