Ion channel targeting within neuronal and muscle membranes is an important determinant of electrical excitability. Recent evidence suggests that there exists within the membrane specialized microdomains commonly referred to as lipid rafts. These domains are enriched in cholesterol and sphingolipids and concentrate a number of signal transduction proteins such as nitricoxide synthase, ligand-gated receptors, and multiple protein kinases. Here, we demonstrate that the voltagegated K ؉ channel Kv2.1, but not Kv4.2, targets to lipid rafts in both heterologous expression systems and rat brain. The Kv2.1 association with lipid rafts does not appear to involve caveolin. Depletion of cellular cholesterol alters the buoyancy of the Kv2.1 associated rafts and shifts the midpoint of Kv2.1 inactivation by nearly 40 mV without affecting peak current density or channel activation. The differential targeting of Kv channels to lipid rafts represents a novel mechanism both for the subcellular sorting of K ؉ channels to regions of the membrane rich in signaling complexes and for modulating channel properties via alterations in lipid content.The subcellular localization of ion channels is necessary for proper electrical signaling. In cardiac and skeletal myocytes, ion channels show a differential surface distribution (1, 2). Within the brain, voltage-gated K ϩ (Kv) channels often show not only polarized sorting to either axons or dendrites, but also isoform-specific localization within dendrites alone. Thus, there exists specific sorting mechanisms for restricting lateral distribution within a given membrane domain (3). One physiological consequence for such specific localization is that it places various signal transduction molecules near their ion channel substrates (4). Several families of intracellular proteins, PDZs and AKAPs, have been shown to cluster both ion channels and modulatory signaling enzymes. Indeed, great emphasis has been placed on the role of PDZ proteins such as PSD-95 in the targeting and localization of ion channels and neurotransmitter receptors (5). In contrast, the role of membrane lipids in differential targeting and integration of ion channels within the plane of the plasma membrane has not been addressed.Recent advances in the study of cell membrane structure have led to the emerging idea that microdomains exist within the fluid bilayer of the plasma membrane. These dynamic structures, termed lipid rafts, are rich in tightly packed sphingolipids and cholesterol (6). The rafts, which are present in both excitable and non-excitable cells, localize a number of membrane proteins, including multiple signal transduction molecules, while excluding others (7). Different types of rafts are likely to exist based on the presence of specific marker proteins and ultrastructure data (8). Caveolae represent one well studied subpopulation of lipid raft having an invaginated morphology and containing the scaffolding protein, caveolin, which interacts directly with several intracellular proteins (7, 9 -12). Here, we de...

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 Xenopus oocytes was enhanced as the membrane potential was progressively depolarized. The IC 50 was 8.4 ؎ 0.9 M when 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 (IC 50 > 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 thesefindingsindicateacriticalroleforTyr-652involtage-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 cationand -stacking interactions with Tyr-652 and Phe-656 of multiple subunits. HERG1 (1) encodes the pore-forming subunits of channels that conduct the rapid delayed rectifier K ϩ current, I Kr (2, 3). Mutation of HERG is a common cause of inherited long QT syndrome, a disorder of cardiac repolarization that predisposes affected individuals to torsade de pointes arrhythmia and sudden death (4). Acquired long QT syndrome is far more common than inherited long QT syndrome and is most often caused by block of HERG channels as a side effect of treatment with commonly used medications including antiarrhythmic, antihistamine, antibiotic, and psychoactive agents (5, 6). Although rare, treatment with the antimalarial drug chloroquine has also been associated with acquired arrhythmias. Prolonged therapy with chloroquine can lead to electrocardiographic changes including T-wave depression or inversion and prolonged QRS and QT intervals (7,8). Prolonged QT intervals caused by chloroquine can induce torsade de pointes (9, 10). At the cellular level, chloroquine decreases the maximum upstroke velocity due to block of sodium current and prolongs the duration of action potentials due to block of inward rectifier current (I K1 ) and I Kr (11). Elucidating the molecular mechanisms of HERG channel block by chloroquine and other drugs may enable the rational design of new pharmaceuticals devoid of this unwanted side effect.The structural basis of HERG channel block by several potent drugs was recently investigated using alanine-scanning mutagenesis (12). Mutation of several amino acid res...

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 I K1. 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 ionchannel 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 I K1 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.IK1 ͉ ion channel ͉ KCNJ2 ͉ malaria ͉ polyamines

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 receptor's response to voltage. Voltage-dependent modulation of muscarinic receptors has important consequences for cellular signalling in excitable tissues and implications for cardiovascular drug development.Abstract The ability to sense transmembrane voltage is a central feature of many membrane proteins, most notably voltage-gated ion channels. Gating current measurements provide valuable information on protein conformational changes induced by voltage. The recent observation that muscarinic G-protein-coupled receptors (GPCRs) generate gating currents confirms their intrinsic capacity to sense the membrane electrical field. Here, we studied the effect of voltage on agonist activation of M2 muscarinic receptors (M2R) in atrial myocytes and how agonist binding alters M2R gating currents. Membrane depolarization decreased the potency of acetylcholine (ACh), but increased the potency and efficacy of pilocarpine (Pilo), as measured by ACh-activated K + current, I KACh . Voltage-induced conformational changes in M2R were modified in a ligand-selective manner: ACh reduced gating charge displacement while Pilo increased the amount of charge displaced. Thus, these ligands manifest opposite voltage-dependent I KACh modulation and exert opposite effects on M2R gating charge displacement. Finally, mutations in the putative ligand binding site perturbed the movement of the M2R voltage sensor. Our data suggest that changes in voltage induce conformational changes in the ligand binding site that alter the agonist-receptor interaction in a ligand-dependent manner. Voltage-dependent GPCR modulation has important implications for cellular signalling in excitable tissues. Gating current measurement allows for the tracking of subtle conformational changes in the receptor that accompany agonist binding and changes in membrane voltage.

Many common medications block delayed rectifier K(+) channels and prolong the duration of cardiac action potentials. Here we investigate the molecular mechanisms of voltage-dependent block of human ether-a-go-go-related gene (HERG) delayed rectifier K(+) channels expressed in Xenopus laevis oocytes by quinidine, an antiarrhythmic drug, and vesnarinone, a cardiotonic drug. The IC(50) values determined with voltage-clamp pulses to 0 mV were 4.6 microM and 57 microM for quinidine and quinine, respectively. Block of HERG by quinidine (and its isomer quinine) was enhanced by progressive membrane depolarization and accompanied by a negative shift in the voltage dependence of channel activation. As reported previously for other HERG blockers (e.g., MK-499, cisapride, terfenadine, chloroquine), the potency of quinidine was reduced >100-fold by the mutation of key aromatic residues (Y652, F656) located in the S6 domain. Mutations of Y652 eliminated (Y652F) or reversed (Y652A) the voltage dependence of HERG channel block by quinidine and quinine. These quinolines contain a charged N atom that might bond with Y652 by a cation-pi interaction. However, similar changes in the voltage-dependent profile for block of Y652F or Y652A HERG channels were observed with vesnarinone, a cardiotonic drug that is uncharged at physiological pH. Together, these results suggest that voltage-dependent block of HERG results from gating-dependent changes in the orientation of Y652, a critical component of the drug binding site, and not from a transmembrane field effect on a charged drug molecule.