Shetuan Zhang

Mechanism of Block and Identification of the Verapamil Binding Domain to HERG Potassium Channels

Calcium channel antagonists have diverse effects on cardiac electrophysiology. We studied the effects of verapamil, diltiazem, and nifedipine on HERG K+ channels that encode IKr in native heart cells. In our experiments, verapamil caused high-affinity block of HERG current (IC50=143.0 nmol/L), a value close to those reported for verapamil block of L-type Ca2+ channels, whereas diltiazem weakly blocked HERG current (IC50=17.3 micromol/L), and nifedipine did not block HERG current. Verapamil block of HERG channels was use and frequency dependent, and verapamil unbound from HERG channels at voltages near the normal cardiac cell resting potential or with drug washout. Block of HERG current by verapamil was reduced by lowering pHO, which decreases the proportion of drug in the membrane-permeable neutral form. N-methyl-verapamil, a membrane-impermeable, permanently charged verapamil analogue, blocked HERG channels only when applied intracellularly. Verapamil antagonized dofetilide block of HERG channels, which suggests that they may share a common binding site. The C-type inactivation-deficient mutations, Ser620Thr and Ser631Ala, reduced verapamil block, which is consistent with a role for C-type inactivation in high-affinity drug block, although the Ser620Thr mutation decreased verapamil block 20-fold more than the Ser631Ala mutation. Our findings suggest that verapamil enters the cell membrane in the neutral form to act at a site within the pore accessible from the intracellular side of the cell membrane, possibly involving the serine at position 620. Thus, verapamil shares high-affinity HERG channel blocking properties with other class III antiarrhythmic drugs, and this may contribute to its antiarrhythmic mechanism.

Block of HERG potassium channels by the antihistamine astemizole and its metabolites desmethylastemizole and norastemizole.

Electrophysiologic Effects of Astemizole Metabolites. Introduction: The selective H1‐receptor antagonist astemizole (Hismanal) causes acquired long QT syndrome. Astemizole blocks the rapidly activating delayed rectifier K+ current IKr and the human ether‐a go‐go‐related gene (HKRG) K+ channels that underlie it. Astemizole also is rapidly metabolized. The principal metabolite is desmethylastemizole, which retains H1‐receptor antagonist properties, has a long elimination time of 9 to 13 days, and its steady‐state serum concentration exceeds that of astemizole by more than 30‐fold. A second metabolite is norastemizole, which appears in serum in low concentrations following astemizole ingestion and has undergone development as a new antihistamine drug. Our objective in the present work was to study the effects of desmethylastemizole, norastemizole, and astemizole on HERG K+ channels.

Extracellular K+ concentration controls cell surface density of IKr in rabbit hearts and of the HERG channel in human cell lines

Although the modulation of ion channel gating by hormones and drugs has been extensively studied, much less is known about how cell surface ion channel expression levels are regulated. Here, we demonstrate that the cell surface density of both the heterologously expressed K+ channel encoded by the human ether-a-go-go-related gene (HERG) and its native counterpart, the rapidly activating delayed rectifier K+ channel (IKr), in rabbit hearts in vivo is precisely controlled by extracellular K+ concentration ([K+]o) within a physiologically relevant range. Reduction of [K+]o led to accelerated internalization and degradation of HERG channels within hours. Confocal analysis revealed colocalization between HERG and ubiquitin during the process of HERG internalization, and overexpression of ubiquitin facilitated HERG degradation under low [K+]o. The HERG channels colocalized with a marker of multivesicular bodies during internalization, and the internalized HERG channels were targeted to lysosomes. Our results provide the first evidence to our knowledge that the cell surface density of a voltage-gated K+ channel, HERG, is regulated by a biological factor, extracellular K+. Because hypokalemia is known to exacerbate long QT syndrome (LQTS) and Torsades de pointes tachyarrhythmias, our findings provide a potential mechanistic link between hypokalemia and LQTS.

Droperidol lengthens cardiac repolarization due to block of the rapid component of the delayed rectifier potassium current

Droperidol Blocks Cardiac IKr. Introduction: Torsades de pointes have been observed during treatment with droperidol, a butyrophenone neuroleptic agent. Our objectives were (1) to characterize the effects of droperidol on cardiac repolarization and (2) to evaluate effects of droperidol on a major time‐dependent outward potassium current involved in cardiac repolarization (IKr).

Identification of IKr and Its Trafficking Disruption Induced by Probucol in Cultured Neonatal Rat Cardiomyocytes

The human ether-a-go-go-related gene (hERG) encodes a channel that conducts the rapidly activating delayed rectifier K+ current (IKr), which is important for cardiac repolarization. Mutations in hERG reduce IKr and cause congenital long QT syndrome (LQTS). More frequently, common medications can reduce IKr and cause LQTS as a side effect. Protein trafficking abnormalities are responsible for most hERG mutation-related LQTS and are recently recognized as a mechanism for drug-induced LQTS. Whereas hERG trafficking has been studied in recombinant expression systems, there has been no reported study on cardiac IKr trafficking at the protein level. In the present study, we identified that IKr is present in cultured neonatal rat ventricular myocytes and can be robustly recorded using Cs+ as the charge carrier. We further discovered that 4,4′-(isopropylidenedithio)-bis-(2,6-di-t-butylphenol) (probucol), a cholesterol-lowering drug that induces LQTS, disrupted IKr trafficking and prolonged the cardiac action potential duration. Probucol did not directly block IKr. Probucol also disrupted hERG trafficking and did not block hERG channels expressed in human embryonic kidney 293 cells. We conclude that probucol induces LQTS by disrupting ether-a-go-go-related gene trafficking, and that primary culture of neonatal rat cardiomyocytes represents a useful system for studying native IKr trafficking.

GABAA receptor activation and the excitability of nerve terminals in the rat posterior pituitary.

1. The activation of GABAA receptors in nerve terminal membranes gates a Cl‐ channel. Experiments were conducted to determine how the activation of this receptor influences membrane potentials, action potentials and voltage‐activated Na+ and K+ channels. 2. When activation of the GABAA receptor produced only conductance changes and no voltage changes, action potentials changed only slightly. The threshold for action potential generation increased by 15%. GABA reduced the broadening of action potentials caused by high frequency stimulation by only 7%. These results indicate that membrane shunting by GABA‐gated Cl‐ channels plays a relatively minor role. 3. By recording changes in the current through K+ channels in cell‐attached patches, the activation of GABAA receptors was shown to depolarize the nerve terminal membrane from rest by 14 mV. The GABAB receptor agonist baclofen produced no change in resting membrane potential as measured by this same technique. 4. In whole‐terminal recordings under current clamp, with pipettes containing various Cl‐ concentrations, the GABA‐induced depolarization increased with Ecl. The variation with Ecl provided a basis for evaluating the contributions of leak and K+ current in the balance of currents that determines the magnitude of the GABA‐induced depolarization. 5. Based on the GABA‐induced voltage change and an evaluation of the other currents of significance in the relevant voltage range, an estimate was obtained for ECl of ‐48 mV to give an estimate for the intracellular Cl‐ ion concentration of 20 mM. 6. Under conditions allowing both conductance and voltage to change during Cl‐ channel gating, GABA prevented action potential responses to current injection. Comparable depolarizations produced by adjusting a steady holding current also blocked action potential responses. 7. A depolarization from ‐60 to ‐45 mV under voltage clamp inactivated approximately 90% of the Na+ channels and activated a small amount of K+ current. This suggests that inactivation of Na+ channels makes a major contribution to the inhibition of action potentials by GABA. 8. These results are consistent with the hypothesis that GABA inhibits neurosecretion by retarding impulse propagation into the terminal arborization. These results support a depolarization block mechanism for the inhibition of secretion, in which depolarization inactivates Na+ channels sufficiently to block action potentials.

Action potential propagation and propagation block by GABA in rat posterior pituitary nerve terminals.

1. A theoretical model was developed to investigate action potential propagation in posterior pituitary nerve terminals. This model was then used to evaluate the efficacy of depolarizing and shunting GABA responses on action potential propagation. 2. Experimental data obtained from the posterior pituitary with patch clamp techniques were used to derive empirical expressions for the voltage and time dependence of the nerve terminal Na+ and K+ channels. The essential structure employed here was based on anatomical and cable data from the posterior pituitary, and consisted of a long cylindrical axon (diameter, 0.5 mm) with a large spherical swelling (diameter, 4‐21 mm) in the middle. 3. In the absence of an inhibitory conductance, simulated action potentials propagated with high fidelity through the nerve terminal. Swellings could block propagation, but only when sizes exceeded those observed in the posterior pituitary. Adding axonal branches reduced the critical size only slightly. These results suggested that action potentials invade the entire posterior pituitary nerve terminal in the absence of inhibition or depression. 4. The addition of inhibitory conductance to a swelling caused simulated action potentials to fail at the swelling. Depolarizing inhibitory conductances were 1.6 times more effective than shunting inhibitory conductances in blocking propagation. 5. Inhibitory conductances within the range of experimentally observed magnitudes and localized to swellings in the observed range of sizes were too weak to block simulated action potentials. However, twofold enhancement of GABA responses by neurosteroid resulted in currents strong enough to block propagation in realistic swelling sizes. 6. GABA could block simulated propagation without neurosteroid enhancement provided that GABA was present throughout a region in the order of a few hundred micrometres. For this widespread inhibition depolarizing conductance was 2.2 times more effective than shunting conductance. 7. These results imply two modes of propagation block, one resulting from highly localized release of inhibitory transmitter under conditions potentiating GABA responses, and the other resulting from widespread release of GABA in the absence of receptor potentiation. 8. The Na+ channels of the posterior pituitary nerve terminal have a unique voltage dependence that allows small depolarizations to inactivate without causing activation. The voltage dependence of this Na+ channel may serve as a specialized adaptation that facilitates in allowing small depolarizing conductances to block action potential propagation.

Molecular determinants of the inhibition of human Kv1.5 potassium currents by external protons and Zn2

Using human Kv1.5 channels expressed in HEK293 cells we assessed the ability of H+o to mimic the previously reported action of Zn2+ to inhibit macroscopic hKv1.5 currents, and using site‐directed mutagenesis, we addressed the mechanistic basis for the inhibitory effects of H+o and Zn2+. As with Zn2+, H+o caused a concentration‐dependent, K+o‐sensitive and reversible reduction of the maximum conductance (gmax). With zero, 5 and 140 mm K+o the pKH for this decrease of gmax was 6.8, 6.2 and 6.0, respectively. The concentration dependence of the block relief caused by increasing [K+]o was well fitted by a non‐competitive interaction between H+o and K+o, for which the KD for the K+ binding site was 0.5‐1.0 mm. Additionally, gating current analysis in the non‐conducting mutant hKv1.5 W472F showed that changing from pH 7.4 to pH 5.4 did not affect Qmax and that charge immobilization, presumed to be due to C‐type inactivation, was preserved at pH 5.4. Inhibition of hKv1.5 currents by H+o or Zn2+ was substantially reduced by a mutation either in the channel turret (H463Q) or near the pore mouth (R487V). In light of the requirement for R487, the homologue of Shaker T449, as well as the block‐relieving action of K+o, we propose that H+ or Zn2+ binding to histidine residues in the pore turret stabilizes a channel conformation that is most likely an inactivated state.

Molecular Determinants of Cocaine Block of Human Ether-á-go-go-Related Gene Potassium Channels

The use of cocaine causes cardiac arrhythmias and sudden death. Blockade of the cardiac potassium channel human ether-á-go-go-related gene (hERG) has been implicated as a mechanism for the proarrhythmic action of cocaine. hERG encodes the pore-forming subunits of the rapidly activating delayed rectifier K+ channel (IKr), which is important for cardiac repolarization. Blockade of IKr/hERG represents a common mechanism for drug-induced long QT syndrome. The mechanisms for many common drugs to block the hERG channel are not well understood. We investigated the molecular determinants of hERG channels in cocaine-hERG interactions using site-targeted mutations and patch-clamp method. Wild-type and mutant hERG channels were heterologously expressed in human embryonic kidney 293 cells. We found that there was no correlation between inactivation gating and cocaine block of hERG channels. We also found that consistent with Thr-623, Tyr-652, and Phe-656 being critical for drug binding to hERG channels, mutations in these residues significantly reduced cocaine-induced block, and the hydrophobicity of the residues at position 656 dictated the cocaine sensitivity of the channel. Although the S620T mutation, which removed hERG inactivation, reduced cocaine block by 21-fold, the S620C mutation, which also completely removed hERG inactivation, did not affect the blocking potency of cocaine. Thus, Ser-620 is another pore helix residue whose mutation can interfere with cocaine binding independently of its effect on inactivation.

Cell Surface Expression of Human Ether-a-go-go-related Gene (hERG) Channels Is Regulated by Caveolin-3 Protein via the Ubiquitin Ligase Nedd4-2

Background: Alterations in hERG-encoded K+ channel current can cause fatal cardiac electrical disturbances. Results: Caveolin-3 enhances ubiquitin ligase Nedd4-2 interaction with mature hERG channels in the plasma membrane, leading to decreased channel expression. Conclusion: Caveolin-3 regulates hERG expression and thus function via Nedd4-2. Significance: Understanding of hERG regulation pathway is important for cardiac electrophysiology and antiarrhythmic strategies. The human ether-a-go-go-related gene (hERG) encodes the rapidly activating delayed rectifier potassium channel (IKr) which plays an important role in cardiac repolarization. A reduction or increase in hERG current can cause long or short QT syndrome, respectively, leading to fatal cardiac arrhythmias. The channel density in the plasma membrane is a key determinant of the whole cell current amplitude. To gain insight into the molecular mechanisms for the regulation of hERG density at the plasma membrane, we used whole cell voltage clamp, Western blotting, and immunocytochemical methods to investigate the effects of an integral membrane protein, caveolin-3 (Cav3) on hERG expression levels. Our data demonstrate that Cav3, hERG, and ubiquitin-ligase Nedd4-2 interact with each other and form a complex. Expression of Cav3 thus enhances the hERG-Nedd4-2 interaction, leading to an increased ubiquitination and degradation of mature, plasma-membrane localized hERG channels. Disrupting Nedd4-2 interaction with hERG by mutations eliminates the effects of Cav3 on hERG channels. Knockdown of endogenous Cav3 or Nedd4-2 in cultured neonatal rat ventricular myocytes using siRNA led to an increase in native IKr. Our data demonstrate that hERG expression in the plasma membrane is regulated by Cav3 via Nedd4-2. These findings extend our understanding of the regulation of hERG channels and cardiac electrophysiology.