The voltage-sensitive cardiac M2 muscarinic receptor modulates the inward rectification of the G protein-coupled, ACh-gated K+ current.

The acetylcholine (ACh)-gated inwardly rectifying K+ current (IKACh) plays a vital role in cardiac excitability by regulating heart rate variability and vulnerability to atrial arrhythmias. These crucial physiological contributions are determined principally by the inwardly rectifying nature of IKACh. Here, we investigated the relative contribution of two distinct mechanisms of IKACh inward rectification measured in atrial myocytes: a rapid component due to KACh channel block by intracellular Mg2+ and polyamines; and a time- and concentration-dependent mechanism. The time- and ACh concentration-dependent inward rectification component was eliminated when IKACh was activated by GTPγS, a compound that bypasses the muscarinic-2 receptor (M2R) and directly stimulates trimeric G proteins to open KACh channels. Moreover, the time-dependent component of IKACh inward rectification was also eliminated at ACh concentrations that saturate the receptor. These observations indicate that the time- and concentration-dependent rectification mechanism is an intrinsic property of the receptor, M2R; consistent with our previous work demonstrating that voltage-dependent conformational changes in the M2R alter the receptor affinity for ACh. Our analysis of the initial and time-dependent components of IKACh indicate that rapid Mg2+-polyamine block accounts for 60-70% of inward rectification, with M2R voltage sensitivity contributing 30-40% at sub-saturating ACh concentrations. Thus, while both inward rectification mechanisms are extrinsic to the KACh channel, to our knowledge, this is the first description of extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of a G protein-coupled receptor.

Pharmacological Conversion of a Cardiac Inward Rectifier into an Outward Rectifier Potassium Channel.

Potassium (K(+)) channels are crucial for determining the shape, duration, and frequency of action-potential firing in excitable cells. Broadly speaking, K(+) channels can be classified based on whether their macroscopic current outwardly or inwardly rectifies, whereby rectification refers to a change in conductance with voltage. Outwardly rectifying K(+) channels conduct greater current at depolarized membrane potentials, whereas inward rectifier channels conduct greater current at hyperpolarized membrane potentials. Under most circumstances, outward currents through inwardly rectifying K(+) channels are reduced at more depolarized potentials. However, the acetylcholine-gated K(+) channel (KACh) conducts current that inwardly rectifies when activated by some ligands (such as acetylcholine), and yet conducts current that outwardly rectifies when activated by other ligands (for example, pilocarpine and choline). The perplexing and paradoxical behavior of KACh channels is due to the intrinsic voltage sensitivity of the receptor that activates KACh channels, the M2 muscarinic receptor (M2R). Emerging evidence reveals that the affinity of M2R for distinct ligands varies in a voltage-dependent and ligand-specific manner. These intrinsic receptor properties determine whether current conducted by KACh channels inwardly or outwardly rectifies. This review summarizes the most recent concepts regarding the intrinsic voltage sensitivity of muscarinic receptors and the consequences of this intriguing behavior on cardiac physiology and pharmacology of KACh channels.

The agonist-specific voltage dependence of M2 muscarinic receptors modulates the deactivation of the acetylcholine-gated K(+) current (I KACh).

Recently, it has been shown that G protein-coupled receptors (GPCRs) display intrinsic voltage sensitivity. We reported that the voltage sensitivity of M2 muscarinic receptor (M2R) is also ligand specific. Here, we provide additional evidence to understand the mechanism underlying the ligand-specific voltage sensitivity of the M2R. Using ACh, pilocarpine (Pilo), and bethanechol (Beth), we evaluated the agonist-specific effects of voltage by measuring the ACh-activated K(+) current (I KACh) in feline and rabbit atrial myocytes and in HEK-293 cells expressing M2R-Kir3.1/Kir3.4. The activation of I KACh by the muscarinic agonist Beth was voltage insensitive, suggesting that the voltage-induced conformational changes in M2R do not modify its affinity for this agonist. Moreover, deactivation of the Beth-evoked I KACh was voltage insensitive. By contrast, deactivation of the ACh-induced I KACh was significantly slower at -100 mV than at +50 mV, while an opposite effect was observed when I KACh was activated by Pilo. These findings are consistent with the voltage affinity pattern observed for these three agonists. Our findings suggest that independent of how voltage disturbs the receptor binding site, the voltage dependence of the signaling pathway is ultimately determined by the agonist. These observations emphasize the pharmacological potential to regulate the M2R-parasympathetic associated cardiac function and also other cellular signaling pathways by exploiting the voltage-dependent properties of GPCRs.

The principal conductance in Giardia lamblia trophozoites possesses functional properties similar to the mammalian ClC-2 current.

The human intestinal pathogen Giardia lamblia is a flagellated unicellular protozoan with pronounced medical and biological relevance. However, the basic physiology of Giardia trophozoites has been sparsely studied, especially the electrical and ionic properties of their cellular membrane which are virtually unknown. In this study, we were able to record and characterize the macroscopic ionic currents of Giardia trophozoite membrane by electrophysiological methods of the patch clamp technique. Giardia trophozoites showed a high current density (∼600 pA/pF at -140 mV) that was activated upon hyperpolarization. This current was carried by a chloride-selective channel (I Cl-G) and it was the most important determinant of the membrane potential in Giardia trophozoites. Moreover, this conductance was able to carry other halide anions and the sequence of permeability was Br(-) > Cl(-) ≈ I(-) ≫ F(-). Besides the voltage-dependent inward-rectifying nature of I Cl-G, its activation and deactivation kinetics were comparable to those observed in ClC-2 channels. Extracellular pH modified the voltage-dependent properties of I Cl-G, shifting the activation curve from a V 1/2 = -79 ± 1 mV (pH 7.4) to -93 ± 2 mV (pH 8.4) and -112 ± 2 mV (pH 5.4). Furthermore, the maximal amplitude of I Cl-G measured at -100 mV showed dependence to external pH in a bell-shaped fashion reported only for ClC-2 channels. Therefore, our results suggest that I Cl-G possesses several functional properties similar to the mammalian ClC-2 channels.

Voltage sensitivity of M2 muscarinic receptors underlies the delayed rectifier-like activation of ACh-gated K(+) current by choline in feline atrial myocytes.

Choline (Ch) is a precursor and metabolite of the neurotransmitter acetylcholine (ACh). In canine and guinea pig atrial myocytes, Ch was shown to activate an outward K(+) current in a delayed rectifier fashion. This current has been suggested to modulate cardiac electrical activity and to play a role in atrial fibrillation pathophysiology. However, the exact nature and identity of this current has not been convincingly established. We recently described the unique ligand- and voltage-dependent properties of muscarinic activation of ACh-activated K(+) current (IKACh) and showed that, in contrast to ACh, pilocarpine induces a current with delayed rectifier-like properties with membrane depolarization. Here, we tested the hypothesis that Ch activates IKACh in feline atrial myocytes in a voltage-dependent manner similar to pilocarpine. Single-channel recordings, biophysical profiles, specific pharmacological inhibition and computational data indicate that the current activated by Ch is IKACh. Moreover, we show that membrane depolarization increases the potency and efficacy of IKACh activation by Ch and thus gives the appearance of a delayed rectifier activating K(+) current at depolarized potentials. Our findings support the emerging concept that IKACh modulation is both voltage- and ligand-specific and reinforce the importance of these properties in understanding cardiac physiology.

Molecular mechanisms of chloroquine inhibition of heterologously expressed Kir6.2/SUR2A channels.

Chloroquine and related compounds can inhibit inwardly rectifying potassium channels by multiple potential mechanisms, including pore block and allosteric effects on channel gating. Motivated by reports that chloroquine inhibition of cardiac ATP-sensitive inward rectifier K(+) current (I(KATP)) is antifibrillatory in rabbit ventricle, we investigated the mechanism of chloroquine inhibition of ATP-sensitive potassium (K(ATP)) channels (Kir6.2/SUR2A) expressed in human embryonic kidney 293 cells, using inside-out patch-clamp recordings. We found that chloroquine inhibits the Kir6.2/SUR2A channel by interacting with at least two different sites and by two mechanisms of action. A fast-onset effect is observed at depolarized membrane voltages and enhanced by the N160D mutation in the central cavity, probably reflecting direct channel block resulting from the drug entering the channel pore from the cytoplasmic side. Conversely, a slow-onset, voltage-independent inhibition of I(KATP) is regulated by chloroquine interaction with a different site and probably involves disruption of interactions between Kir6.2/SUR2A and phosphatidylinositol 4,5-bisphosphate. Our findings reveal multiple mechanisms of K(ATP) channel inhibition by chloroquine, highlighting the numerous convergent regulatory mechanisms of these ligand-dependent ion channels.

Differential voltage-dependent modulation of the ACh-gated K+ current by adenosine and acetylcholine.

Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the 'relaxation' property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property.

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

Normal heart rate variability is critically dependent upon the G-protein-coupled, acetylcholine (ACh)-activated inward rectifier K+ current, I(KACh). A unique feature of I(KACh) is the so-called 'relaxation' gating property that contributes to increased current at hyperpolarized membrane potentials. I(KACh) relaxation refers to a slow decrease or increase in current magnitude with depolarization or hyperpolarization, respectively. The molecular mechanism underlying this perplexing gating behaviour remains unclear. Here, we consider a novel explanation for I(KACh) relaxation based upon the recent finding that G-protein-coupled receptors (GPCRs) are intrinsically voltage sensitive and that the muscarinic agonists acetylcholine (ACh) and pilocarpine (Pilo) manifest opposite voltage-dependent I(KACh) modulation. We show that Pilo activation of I(KACh) displays relaxation characteristics opposite to that of ACh. We explain the opposite effects of ACh and Pilo using Markov models of I(KACh) that incorporate ligand-specific, voltage-dependent parameters. Based on experimental and computational findings, we propose a novel molecular mechanism to describe the enigmatic relaxation gating process: I(KACh) relaxation represents a voltage-dependent change in agonist affinity as a consequence of a voltage-dependent conformational change in the muscarinic receptor.

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

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 agonist­receptor 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.

Voltage-dependent potassium currents in feline sino-atrial node myocytes.

We characterized the properties of the voltage-dependent K(+) currents I (to), I (Kr), and I (Ks) in isolated feline sino-atrial node (SAN) myocytes. I (to) activated rapidly and then inactivated with a single exponential and voltage-independent time course. Recovery from inactivation of I (to) followed a single exponential time course with τ = 21.1 ± 2.5 ms, at -80 mV. Steady-state inactivation relationship showed a V½ of inactivation at -47.9 ± 2.3 mV. These biophysical properties are similar to the fast I (to) phenotype of other mammals. I (Kr) exhibited typical negative slope conductance at test potentials > 0 mV and slow deactivation. I (Ks) activated very slowly. The functional contribution of I (to), I (Kr), and I (Ks) to the sustained pacemaking activity of feline SAN myocytes was analyzed. Similar to other mammals, I (to) underlies the initial repolarization phase of the SAN action potential, whereas I (Kr) and I (Ks) mediate repolarization back to the maximal diastolic potential. I (Kr) and I (Ks) also contribute to diastolic depolarization because of their slow deactivation kinetics. The I (Kr) specific blocker E-4031 and the I (Ks) blocker HMR 1556 significantly increased action potential duration, but had negligible effects on the maximum diastolic potential and only modest effects on the frequency of spontaneous activity, suggesting that each one of these two currents itself is capable of supporting action potential repolarization in the feline sinus node.

Muscarinic-activated potassium current mediates the negative chronotropic effect of pilocarpine on the rabbit sinoatrial node.

Pilocarpine is a nonspecific agonist of muscarinic receptors which was recently found to activate the M(2) receptor subtype in a voltage-dependent manner. The purpose of our study was to investigate the role of the acetylcholine (muscarinic)-activated K(+) current (I (KACh)) on the negative chronotropic effect of pilocarpine in rabbit sinoatrial node. In multicellular preparations, we studied the effect of pilocarpine on spontaneous action potentials. In isolated myocytes, using the patch clamp technique, we studied the effects of pilocarpine on I (KACh). Pilocarpine produced a decrease in spontaneous frequency, hyperpolarization of the maximum diastolic potential, and a decrease in the diastolic depolarization rate. These effects were partially antagonized by tertiapin Q. Cesium and calyculin A in the presence of tertiapin Q partially prevented the effects of pilocarpine. In isolated myocytes, pilocarpine activated the muscarinic potassium current, I (KACh) in a voltage-dependent manner. In conclusion, the negative chronotropic effects of pilocarpine on the sinatrial node could be mainly explained by activation of I (KACh).

Mechanisms for Kir channel inhibition by quinacrine: acute pore block of Kir2.x channels and interference in PIP2 interaction with Kir2.x and Kir6.2 channels.

Cardiac inward rectifier potassium currents determine the resting membrane potential and contribute repolarization capacity during phase 3 repolarization. Quinacrine is a cationic amphiphilic drug. In this work, the effects of quinacrine were studied on cardiac Kir channels expressed in HEK 293 cells and on the inward rectifier potassium currents, I(K1) and I(KATP), in cardiac myocytes. We found that quinacrine differentially inhibited Kir channels, Kir6.2 ∼ Kir2.3 > Kir2.1. In addition, we found in cardiac myocytes that quinacrine inhibited I(KATP) > I(K1). We presented evidence that quinacrine displays a double action towards strong inward rectifier Kir2.x channels, i.e., direct pore block and interference in phosphatidylinositol 4,5-bisphosphate, PIP(2)-Kir channel interaction. Pore block is evident in Kir2.1 and 2.3 channels as rapid block; channel block involves residues E224 and E299 facing the cytoplasmic pore of Kir2.1. The interference of the drug with the interaction of Kir2.x and Kir6.2/SUR2A channels and PIP(2) is suggested from four sources of evidence: (1) Slow onset of current block when quinacrine is applied from either the inside or the outside of the channel. (2) Mutation of Kir2.3(I213L) and mutation of Kir6.2(C166S) increase their affinity for PIP(2) and lowers its sensitivity for quinacrine. (3) Mutations of Kir2.1(L222I and K182Q) which decreased its affinity for PIP(2) increased its sensitivity for quinacrine. (4) Co-application of quinacrine with PIP(2) lowers quinacrine-mediated current inhibition. In conclusion, our data demonstrate how an old drug provides insight into a dual a blocking mechanism of Kir carried inward rectifier channels.

The antimalarial drug mefloquine inhibits cardiac inward rectifier K+ channels: evidence for interference in PIP2-channel interaction.

The antimalarial drug mefloquine was found to inhibit the KATP channel by an unknown mechanism. Because mefloquine is a Cationic amphiphilic drug and is known to insert into lipid bilayers, we postulate that mefloquine interferes with the interaction between PIP2 and Kir channels resulting in channel inhibition. We studied the inhibitory effects of mefloquine on Kir2.1, Kir2.3, Kir2.3(I213L), and Kir6.2/SUR2A channels expressed in HEK-293 cells, and on IK1 and IKATP from feline cardiac myocytes. The order of mefloquine inhibition was Kir6.2/SUR2A ≈ Kir2.3 (IC50 ≈ 2 µM) > Kir2.1 (IC50 > 30 µM). Similar results were obtained in cardiac myocytes. The Kir2.3(I213L) mutant, which enhances the strength of interaction with PIP2 (compared to WT), was significantly less sensitive (IC50 = 9 µM). In inside-out patches, continuous application of PIP2 strikingly prevented the mefloquine inhibition. Our results support the idea that mefloquine interferes with PIP2-Kir channels interactions.

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 I(K1) 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 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 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.

Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism.

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg2+. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K+-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K+-flux is convergent with the gating of PIP2 because, at a saturating concentration, PIP2 greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K+-flux gating.

Riluzole inhibits Kv4.2 channels acting on the closed and closed inactivated states.

Riluzole is an anticonvulsant drug also used to treat the amyotrophic lateral sclerosis and major depressive disorder. This compound has antiglutamatergic activity and is an important multichannel blocker. However, little is known about its actions on the Kv4.2 channels, the molecular correlate of the A-type K+ current (IA) and the fast transient outward current (Itof). Here, we investigated the effects of riluzole on Kv4.2 channels transiently expressed in HEK-293 cells. Riluzole inhibited Kv4.2 channels with an IC50 of 190 ± 14 µM and the effect was voltage- and frequency-independent. The activation rate of the current (at +50 mV) was not affected by the drug, nor the voltage dependence of channel activation, but the inactivation rate was accelerated by 100 and 300 µM riluzole. When Kv4.2 channels were maintained at the closed state, riluzole incubation induced a tonic current inhibition. In addition, riluzole significantly shifted the voltage dependence of inactivation to hyperpolarized potentials without affecting the recovery from inactivation. In the presence of the drug, the closed-state inactivation was significantly accelerated, and the percentage of inactivated channels was increased. Altogether, our findings indicate that riluzole inhibits Kv4.2 channels mainly affecting the closed and closed-inactivated states.

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 (IC(50) approximately 200 nM) from the cytoplasmic membrane surface, consistent with open-channel block. Alanine-scanning mutagenesis revealed that AEA interacts with two crucial beta-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.

Multiple effects of 4-aminopyridine on feline and rabbit sinoatrial node myocytes and multicellular preparations.

4-aminopyridine (4-AP) is commonly used to block the transient outward potassium current, I(to), in cardiac and noncardiac tissues. In the present work, we found that 4-AP inhibited the rapid component of the delayed rectifier potassium current, I(Kr), in rabbit-isolated sinoatrial node myocytes by 25% (1 mM) and 51% (5 mM) and inhibited the slow component of the delayed rectifier potassium current, I(Ks), in cat- isolated sinoatrial node myocytes by 39% (1 mM) and 62% (5 mM). In cat- and rabbit-isolated sinoatrial node myocytes, 4-AP activated muscarinic receptors in a voltage-dependent manner to increase the acetylcholine-activated potassium current, I(KACh). In multicellular preparations of the central region of the sinoatrial node from nonreserpinized rabbits, 4-AP produced an increase in action potential overshoot, frequency, and rate of diastolic depolarization. In the presence of the beta-adrenergic antagonist propranolol, 4-AP produced a marked increase in duration and a marked decrease in maximum diastolic potential and eventually, cessation of the spontaneous activity in preparations from the sinoatrial central region. In multicellular preparations from reserpinized rabbits, 4-AP produced similar effects to those observed in the presence of propranolol. We conclude that 4-AP inhibits multiple cardiac K(+) currents, including I(to), I(Kr), and I(Ks), and that these activities mask I(KACh) activation. In addition, in multicellular preparations, 4-AP produces neurotransmitter release from the autonomic nerve terminals. These multiple effects need to be considered when using 4-AP as a "specific" I(to) blocker.

Tamoxifen inhibits cardiac ATP-sensitive and acetylcholine-activated K+ currents in part by interfering with phosphatidylinositol 4,5-bisphosphate-channel interaction.

Tamoxifen inhibits transmembrane currents of the Kir2.x inward rectifier potassium channels by interfering with the interaction of the channels with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)). We tested the hypothesis that Kir channels with low affinity for PIP(2), like the adenosine triphosphate (ATP)-sensitive K(+) channel (K(ATP)) and acetylcholine (ACh)-activated K(+) channel (K(ACh)), have at least the same sensitivity to tamoxifen as Kir2.3. We investigated the effects of tamoxifen (0.1 - 10 microM) on Kir6.2/SUR2A (K(ATP)) and Kir3.1/3.4 (K(ACh)) channels expressed in HEK-293 cells and ATP-sensitive K(+) current (I(KATP)) and ACh-activated K(+) current (I(KACh)) in feline atrial myocytes. The onset of tamoxifen inhibition of both I(KATP) and I(KACh) was slow (T(1/2) approximately 3.5 min) and concentration-dependent but voltage-independent. The time course and degree of inhibition was independent of external or internal drug application. Tamoxifen interacts with the pore forming subunit, Kir6.2, rather than with the SUR subunit. The inhibitory potency of tamoxifen on the Kir6.2/SUR2A channel was decreased by the mutation (C166S) on Kir6.2 and in the continuous presence of PIP(2). In atrial myocytes, the mechanism and potency of the effects of tamoxifen on K(ATP) and K(ACh) channels were comparable to those in HEK-293 cells. These data suggest that, similar to its effects on Kir2.x currents, tamoxifen inhibits K(ATP) and K(ACh) currents by interfering with the interaction between the channel and PIP(2).

Chloroquine blocks a mutant Kir2.1 channel responsible for short QT syndrome and normalizes repolarization properties in silico.

Short QT Syndrome (SQTS) is a novel clinical entity characterized by markedly rapid cardiac repolarization and lethal arrhythmias. A mutation in the Kir2.1 inward rectifier K+ channel (D172N) causes one form of SQTS (SQT3). Pharmacologic block of Kir2.1 channels may hold promise as potential therapy for SQT3. We recently reported that the anti-malarial drug chloroquine blocks Kir2.1 channels by plugging the cytoplasmic pore domain. In this study, we tested whether chloroquine blocks D172N Kir2.1 channels in a heterologous expression system and if chloroquine normalizes repolarization properties using a mathematical model of a human ventricular myocyte. Chloroquine caused a dose- and voltage-dependent reduction in wild-type (WT), D172N and WT-D172N heteromeric Kir2.1 current. The potency and kinetics of chloroquine block of D172N and WT-D172N Kir2.1 current were similar to WT. In silico modeling of the heterozygous WT-D172N Kir2.1 condition predicted that 3 microM chloroquine normalized inward rectifier K+ current magnitude, action potential duration and effective refractory period. Our results suggest that therapeutic concentrations of chloroquine might lengthen cardiac repolarization in SQT3.