Ricardo A. Navarro-Polanco

Molecular determinants of voltage-dependent human ether-a-go-go related gene (HERG) K+ channel block.

The structural determinants for the voltage-dependent block of ion channels are poorly understood. Here we investigate the voltage-dependent block of wild-type and mutant human ether-a-go-go related gene (HERG) K+ channels by the antimalarial compound chloroquine. The block of wild-type HERG channels expressed in Xenopusoocytes was enhanced as the membrane potential was progressively depolarized. The IC50 was 8.4 ± 0.9 μmwhen assessed during 4-s voltage clamp pulses to 0 mV. Chloroquine also slowed the apparent rate of HERG deactivation, reflecting the inability of drug-bound channels to close. Mutation to alanine of aromatic residues (Tyr-652 or Phe-656) located in the S6 domain of HERG greatly reduced the potency of channel block by chloroquine (IC50 > 1 mm at 0 mV). However, mutation of Tyr-652 also altered the voltage dependence of the block. In contrast to wild-type HERG, block of Y652A HERG channels was diminished by progressive membrane depolarization, and complete relief from block was observed at +40 mV. HERG channel block was voltage-independent when the hydroxyl group of Tyr-652 was removed by mutating the residue to Phe. Together these findings indicate a critical role for Tyr-652 in voltage-dependent block of HERG channels. Molecular modeling was used to define energy-minimized dockings of chloroquine to the central cavity of HERG. Our experimental findings and modeling suggest that chloroquine preferentially blocks open HERG channels by cation-π and π-stacking interactions with Tyr-652 and Phe-656 of multiple subunits.

The molecular basis of chloroquine block of the inward rectifier Kir2.1 channel

Although chloroquine remains an important therapeutic agent for treatment of malaria in many parts of the world, its safety margin is very narrow. Chloroquine inhibits the cardiac inward rectifier K+ current IK1 and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiac IK1. The voltage- and K+-dependence of chloroquine block implied that the binding site was located within the ion-conduction pathway. Site-directed mutagenesis revealed the location of the chloroquine-binding site within the cytoplasmic pore domain rather than within the transmembrane pore. Molecular modeling suggested that chloroquine blocks Kir2.1 channels by plugging the cytoplasmic conduction pathway, stabilized by negatively charged and aromatic amino acids within a central pocket. Unlike most ion-channel blockers, chloroquine does not bind within the transmembrane pore and thus can reach its binding site, even while polyamines remain deeper within the channel vestibule. These findings explain how a relatively low-affinity blocker like chloroquine can effectively block IK1 even in the presence of high-affinity endogenous blockers. Moreover, our findings provide the structural framework for the design of safer, alternative compounds that are devoid of Kir2.1-blocking properties.

RNA editing modulates the binding of drugs and highly unsaturated fatty acids to the open pore of Kv potassium channels

The time course of inactivation of voltage‐activated potassium (Kv) channels is an important determinant of the firing rate of neurons. In many Kv channels highly unsaturated lipids as arachidonic acid, docosahexaenoic acid and anandamide can induce fast inactivation. We found that these lipids interact with hydrophobic residues lining the inner cavity of the pore. We analysed the effects of these lipids on Kv1.1 current kinetics and their competition with intracellular tetraethylammonium and Kvβ subunits. Our data suggest that inactivation most likely represents occlusion of the permeation pathway, similar to drugs that produce ‘open‐channel block’. Open‐channel block by drugs and lipids was strongly reduced in Kv1.1 channels whose amino acid sequence was altered by RNA editing in the pore cavity, and in Kv1.x heteromeric channels containing edited Kv1.1 subunits. We show that differential editing of Kv1.1 channels in different regions of the brain can profoundly alter the pharmacology of Kv1.x channels. Our findings provide a mechanistic understanding of lipid‐induced inactivation and establish RNA editing as a mechanism to induce drug and lipid resistance in Kv channels.

Conformational changes in the M2 muscarinic receptor induced by membrane voltage and agonist binding

Non‐technical summary  Muscarinic receptors were recently shown to be modulated by membrane potential. Here, we show that membrane potential alters the binding of agonists in an agonist‐specific manner. Moreover, agonist binding results in agonist‐specific conformational changes in the muscarinic receptor, as measured by changes in the receptors response to voltage. Voltage‐dependent modulation of muscarinic receptors has important consequences for cellular signalling in excitable tissues and implications for cardiovascular drug development.

4‐aminopyridine activates potassium currents by activation of a muscarinic receptor in feline atrial myocytes.

1. The effects of 4‐aminopyridine (4‐AP) on action potentials, macroscopic membrane currents and single‐channel recording from cardiac left atrial myocytes of the adult cat were studied using the whole‐cell and cell‐attached configurations of the patch‐clamp technique. 2. 4‐AP (1 mM) produced a hyperpolarization of the resting membrane potential and a shortening of action potential duration. Under voltage‐clamp conditions, we have found that 4‐AP increased a background current and a delayed rectifier outward current. These effects were antagonized by atropine. In addition, both effects seemed to be mediated through a pertussis toxin‐sensitive G protein. 3. The background current induced by 4‐AP displayed properties that are highly similar to those of the inwardly rectifying potassium current activated by acetylcholine (IK(ACh)). The time‐dependent potassium current activated by 4‐AP has kinetic and pharmacological properties different from those of the delayed rectifier potassium current previously identified in cardiac myocytes. 4. The activation of the delayed rectifier‐like potassium current could be explained by the activation of a novel muscarinic receptor subtype in which acetylcholine acts as the antagonist. Another possibility is that 4‐AP activates IK(ACh) in a time‐ and voltage‐dependent manner.

Block of hERG channels by berberine : Mechanisms of voltage- and state-dependence probed with site-directed mutant channels

Berberine prolongs the duration of cardiac action potentials without affecting resting membrane potential or action potential amplitude. Controversy exists regarding whether berberine exerts this action by preferential block of different components of the delayed rectifying potassium current, IKr and IKs. Here we have studied the effects of berberine on hERG (IKr) and KCNQ1/KCNE1 (IKs) channels expressed in HEK-293 cells and Xenopus oocytes. In HEK-293 cells, the IC50 for berberine was 3.1 ± 0.5 μM on hERG compared with 11 ± 4% decreases on KCNQ1/KCNE1 channels by 100 μM berberine. Likewise in oocytes, hERG channels were more sensitive to block by berberine (IC50 = 80 ± 5 μM) compared with KCNQ1/KCNE1 channels (∼20% block at 300 μM). hERG block was markedly increased by membrane depolarization. Mutation to Ala of Y652 or F656 located on the S6 domain, or V625 located at the base of the pore helix of hERG decreased sensitivity to block by berberine. An inactivation-deficient mutant hERG channel (G628C/S631C) was also blocked by berberine. Together these findings indicate that berberine preferentially blocks the open state of hERG channels by interacting with specific residues that were previously reported to be important for binding of more potent antagonists.

Relaxation gating of the acetylcholine-activated inward rectifier K + current is mediated by intrinsic voltage sensitivity of the muscarinic receptor

Non‐technical summary  Normal heart rate variability is critically dependent upon the G‐protein‐coupled, acetylcholine (ACh)‐activated inward rectifier K+ current, IKACh. A unique feature of IKACh is the so‐called ‘relaxation’ gating property that contributes to increased current at hyperpolarized membrane potentials. Here, we consider a novel explanation for IKACh relaxation based upon the recent finding that G‐protein‐coupled receptors are intrinsically voltage sensitive and that the muscarinic agonists acetylcholine and pilocarpine manifest opposite voltage‐dependent IKACh modulation. Based on experimental and computational findings, we propose that IKACh relaxation represents a voltage‐dependent change in agonist affinity as a consequence of a voltage‐dependent conformational change in the muscarinic receptor.

Block of wild-type and inactivation-deficient human ether-a-go-go-related gene K+ channels by halofantrine

Halofantrine is an antimalarial drug developed as a treatment of P. falciparum resistant to chloroquine. However, halofantrine can also induce long QT syndrome (LQTS) and torsades de pointes, a potentially life-threatening ventricular arrhythmia. Drug-induced LQTS is usually caused by block of the human ether-a-go-go-related gene (HERG) channels that conduct the rapid delayed rectifier K+ current, IKr, in the heart. Here we show that halofantrine preferentially blocks open and inactivated HERG channels heterologously expressed in Xenopus laevis oocytes. The half-maximal inhibitory concentration (IC50) for block of wild-type (WT) HERG was 1.0 μM. As we reported previously for other HERG channel blockers, the potency of halofantrine was reduced by mutation to Ala of aromatic residues (Y652, F656) located in the S6 domain, or a Val (V625) located in the pore helix. Halofantrine at a concentration 10 μM did not affect the transient outward potassium channel, Kv4.3, the slow delayed rectifier potassium channel, KvLQT1+minK and inward rectifier potassium channel, Kir2.1. An inactivation deficient mutant (G628C/S631C HERG) was only slightly less sensitive (IC50=2.0 μM). The rate of block onset by halofantrine at 0 mV was used to estimate the apparent association (kon) and dissociation (koff) rate constants for drug binding. For WT and G628C/S631C HERG, kon was similar (0.0114 and 0.0163 M−1/s−1 respectively). In contrast, koff was significantly faster for G628C/S631C (0.357 s−1) than WT (0.155 s−1), and explains the observed decrease in drug potency for the inactivation-deficient mutant channel. We conclude that halofantrine requires channels to open before it can gain access to its binding site located in the central cavity of the HERG channel.

Molecular Basis for a High-Potency Open-Channel Block of Kv1.5 Channel by the Endocannabinoid Anandamide

The endocannabinoid, N-arachidonoylethanolamine (anandamide; AEA) is known to interact with voltage-gated K+ (Kv) channels in a cannabinoid receptor-independent manner. AEA modulates the functional properties of Kv channels, converting channels with slowly inactivating current into apparent fast inactivation. In this study, we characterize the mechanism of action and binding site for AEA on Kv1.5 channels expressed on HEK-293 cells using the patch-clamp techniques. AEA exhibited high-potency block (IC50 ≈ 200 nM) from the cytoplasmic membrane surface, consistent with open-channel block. Alanine-scanning mutagenesis revealed that AEA interacts with two crucial β-branching amino acids, Val505 and Ile508 within the S6 domain. Both residues face toward the central cavity and constitute a motif that forms a hydrophobic ring around the ion conduction pathway. This hydrophobic ring motif may be a critical determinant of cannabinoid receptor-independent AEA modulation in other K+ channel families.

Modulation of Cardiac Action Potential and Underlying Ionic Currents by the Pyrethroid Insecticide Deltamethrin

BACKGROUND Organophosphorus, DDT, and pyrethroid insecticides are potent neurotoxic compounds. In addition, serious cardiovascular toxic manifestations such as ventricular arrhythmias have been reported. The objective of the present work was to study possible cardiac electrophysiologic effects of the type II pyrethroid, deltamethrin. METHODS The effects of deltamethrin were studied in enzymatically isolated cat ventricular myocytes. Whole cell, patch-clamp technique was used in current- and voltage-clamp modes. RESULTS Deltamethrin significantly increased action potential duration. Under voltage-clamp conditions, the most striking effects of deltamethrin were on sodium current. Pyrethroid induced a sustained component of the sodium current and caused a negative shift in current-voltage relation for peak current. The drug also slightly inhibited delayed rectifying outward potassium current. CONCLUSIONS Pyrethroid type II, deltamethrin, increased action potential duration due to modulation of sodium current. This effect of deltamethrin can be potentially arrhythmogenic because it can induce Q-T prolongation of ECG and ventricular arrhythmias of the torsade-de-pointes type.