The history and geographic distribution of a KCNQ1 atrial fibrillation risk allele.

The genetic architecture of atrial fibrillation (AF) encompasses low impact, common genetic variants and high impact, rare variants. Here, we characterize a high impact AF-susceptibility allele, KCNQ1 R231H, and describe its transcontinental geographic distribution and history. Induced pluripotent stem cell-derived cardiomyocytes procured from risk allele carriers exhibit abbreviated action potential duration, consistent with a gain-of-function effect. Using identity-by-descent (IBD) networks, we estimate the broad- and fine-scale population ancestry of risk allele carriers and their relatives. Analysis of ancestral migration routes reveals ancestors who inhabited Denmark in the 1700s, migrated to the Northeastern United States in the early 1800s, and traveled across the Midwest to arrive in Utah in the late 1800s. IBD/coalescent-based allele dating analysis reveals a relatively recent origin of the AF risk allele (~5000 years). Thus, our approach broadens the scope of study for disease susceptibility alleles to the context of human migration and ancestral origins.

Dual regulation of hEAG1 channels by phosphatidylinositol 4,5-bisphosphate.

The ether-à-go-go1 (EAG1, Kv10.1) K+ channel is a member of the voltage-gated K+ channel family mainly expressed in the central nervous system and cancer cells. Membrane lipids regulate several voltage-gated K+ channels but their influence on EAG1 channels has been poorly explored. Here we have studied the regulation of hEAG1 channels by phosphatidylinositol 4,5-bisfofate (PIP2) by using different strategies to manipulate the levels of this lipid, and the patch clamp technique. We found that depletion of endogenous PIP2 by activation of the voltage-sensing phosphatase from Danio rerio (Dr-VSP) or the human muscarinic type-1 receptor (hM1R) inhibits hEAG1 currents; however, the application of exogenous PIP2 to increase the level of this lipid on the plasma membrane, also induced an inhibition of hEAG1. In summary, our results indicate that PIP2 have dual effects on hEAG1 channels and its action as activator or inhibitor depends on its initial level on the plasma membrane.

A near-infrared fluorescent voltage-sensitive dye allows for moderate-throughput electrophysiological analyses of human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM)-based assays are emerging as a promising tool for the in vitro preclinical screening of QT interval-prolonging side effects of drugs in development. A major impediment to the widespread use of human iPSC-CM assays is the low throughput of the currently available electrophysiological tools. To test the precision and applicability of the near-infrared fluorescent voltage-sensitive dye 1-(4-sulfanatobutyl)-4-{ß[2-(di-n-butylamino)-6-naphthyl]butadienyl}quinolinium betaine (di-4-ANBDQBS) for moderate-throughput electrophysiological analyses, we compared simultaneous transmembrane voltage and optical action potential (AP) recordings in human iPSC-CM loaded with di-4-ANBDQBS. Optical AP recordings tracked transmembrane voltage with high precision, generating nearly identical values for AP duration (AP durations at 10%, 50%, and 90% repolarization). Human iPSC-CMs tolerated repeated laser exposure, with stable optical AP parameters recorded over a 30-min study period. Optical AP recordings appropriately tracked changes in repolarization induced by pharmacological manipulation. Finally, di-4-ANBDQBS allowed for moderate-throughput analyses, increasing throughput >10-fold over the traditional patch-clamp technique. We conclude that the voltage-sensitive dye di-4-ANBDQBS allows for high-precision optical AP measurements that markedly increase the throughput for electrophysiological characterization of human iPSC-CMs.

ABCC9 is a novel Brugada and early repolarization syndrome susceptibility gene.

BACKGROUND: Genetic defects in KCNJ8, encoding the Kir6.1 subunit of the ATP-sensitive K(+) channel (I(K-ATP)), have previously been associated with early repolarization (ERS) and Brugada (BrS) syndromes. Here we test the hypothesis that genetic variants in ABCC9, encoding the ATP-binding cassette transporter of IK-ATP (SUR2A), are also associated with both BrS and ERS. METHODS AND RESULTS: Direct sequencing of all ERS/BrS susceptibility genes was performed on 150 probands and family members. Whole-cell and inside-out patch-clamp methods were used to characterize mutant channels expressed in TSA201-cells. Eight ABCC9 mutations were uncovered in 11 male BrS probands. Four probands, diagnosed with ERS, carried a highly-conserved mutation, V734I-ABCC9. Functional expression of the V734I variant yielded a Mg-ATP IC50 that was 5-fold that of wild-type (WT). An 18-y/o male with global ERS inherited an SCN5A-E1784K mutation from his mother, who displayed long QT intervals, and S1402C-ABCC9 mutation from his father, who displayed an ER pattern. ABCC9-S1402C likewise caused a gain of function of IK-ATP with a shift of ATP IC50 from 8.5 ± 2 mM to 13.4 ± 5 µM (p<0.05). The SCN5A mutation reduced peak INa to 39% of WT (p<0.01), shifted steady-state inactivation by -18.0 mV (p<0.01) and increased late I(Na) from 0.14% to 2.01% of peak I(Na) (p<0.01). CONCLUSION: Our study is the first to identify ABCC9 as a susceptibility gene for ERS and BrS. Our findings also suggest that a gain-of-function in I(K-ATP) when coupled with a loss-of-function in SCN5A may underlie type 3 ERS, which is associated with a severe arrhythmic phenotype.

The absence of insulin signaling in the heart induces changes in potassium channel expression and ventricular repolarization.

Diabetes mellitus increases the risk for cardiac dysfunction, heart failure, and sudden death. The wide array of neurohumoral changes associated with diabetes pose a challenge to understanding the roles of specific pathways that alter cardiac function. Here, we use a mouse model with cardiomyocyte-restricted deletion of insulin receptors (CIRKO, cardiac-specific insulin receptor knockout) to study the specific effects of impaired cardiac insulin signaling on ventricular repolarization, independent of the generalized metabolic derangements associated with diabetes. Impaired insulin action caused a reduction in mRNA and protein expression of several key K(+) channels that dominate ventricular repolarization. Specifically, components of transient outward K(+) current fast component (Ito,fast; Kv4.2 and KChiP2) were reduced, consistent with a reduction in the amplitude of Ito,fast in isolated left ventricular CIRKO myocytes, compared with littermate controls. The reduction in Ito,fast resulted in ventricular action potential prolongation and prolongation of the QT interval on the surface ECG. These results support the notion that the lack of insulin signaling in the heart is sufficient to cause the repolarization abnormalities described in other animal models of diabetes.

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.

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.

Carvedilol inhibits Kir2.3 channels by interference with PIP2-channel interaction.

Carvedilol, a ß- and α-adrenoceptor blocker, is used to treat congestive heart failure, mild to moderate hypertension, and myocardial infarction. It has been proposed to block K(ATP) channels by binding to the bundle crossing region at a domain including cysteine at position 166, and thereby plugging the pore region. However, carvedilol was reported not to affect Kir2.1 channels, which lack 166 Cys. Here, we demonstrate that carvedilol inhibits Kir2.3 carried current by an alternative mechanism. Carvedilol inhibited Kir2.3 channels with at least 100 fold higher potency (IC(50)=0.49 µM) compared to that for Kir2.1 (IC(50)>50 µM). Kir2.3 channel inhibition was concentration-dependent and voltage-independent. Increasing Kir2.3 channel affinity for PIP(2), by a I213L point mutation, decreased the inhibitory effect of carvedilol more than twentyfold (IC(50)=11.1 µM). In the presence of exogenous PIP(2), Kir2.3 channel inhibition by carvedilol was strongly reduced (80 vs. 2% current inhibition). These results suggest that carvedilol, as other cationic amphiphilic drugs, inhibits Kir2.3 channels by interfering with the PIP(2)-channel interaction.

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.

Structural bases for the different anti-fibrillatory effects of chloroquine and quinidine.

AIMS: Chloroquine, an anti-malarial quinoline, is structurally similar to quinidine. Both drugs have been shown to block ion channels. We tested the hypothesis that chloroquine's mode of interaction with the vestibule of the cytoplasmic domain of the inward rectifier potassium channel Kir2.1 makes it a more effective I(K1) blocker and anti-fibrillatory agent than quinidine. METHODS AND RESULTS: We used comparative molecular modelling and ligand docking of the three-dimensional structures of quinidine and chloroquine in the intracellular domain of Kir2.1. Simulations predicted that chloroquine effectively blocks potassium flow by binding at the centre of the ion permeation vestibule of Kir2.1. In contrast, quinidine binds the vestibular side, only partially blocking ion movement. We tested the modelling predictions in Kir2.1-expressing human embryonic kidney (HEK)-293 cells. The half-maximal inhibitory concentration for chloroquine block of I(K1) was 1.2 µM, while that of quinidine was 57 µM. Finally, we used optical mapping of Langendorff-perfused mouse hearts with cardiac-specific Kir2.1 up-regulation to compare the anti-fibrillatory effects of the drugs. In five of six hearts, 10 µM quinidine slowed the frequency but did not terminate the tachyarrhythmia. In five of five hearts, 10 µM chloroquine terminated the arrhythmia, restoring sinus rhythm. CONCLUSION: Quinidine only partially blocks I(K1). Chloroquine binds at the centre of the ion permeation vestibule of Kir2.1, which makes it a more effective I(K1) blocker and anti-fibrillatory agent than quinidine. Integrating the structural biology of drug-ion channel interactions with cellular electrophysiology and optical mapping is an excellent approach to understand the molecular mechanisms of anti-arrhythmic drug action and for drug discovery.

Flecainide increases Kir2.1 currents by interacting with cysteine 311, decreasing the polyamine-induced rectification.

Both increase and decrease of cardiac inward rectifier current (I(K1)) are associated with severe cardiac arrhythmias. Flecainide, a widely used antiarrhythmic drug, exhibits ventricular proarrhythmic effects while effectively controlling ventricular arrhythmias associated with mutations in the gene encoding Kir2.1 channels that decrease I(K1) (Andersen syndrome). Here we characterize the electrophysiological and molecular basis of the flecainide-induced increase of the current generated by Kir2.1 channels (I(Kir2.1)) and I(K1) recorded in ventricular myocytes. Flecainide increases outward I(Kir2.1) generated by homotetrameric Kir2.1 channels by decreasing their affinity for intracellular polyamines, which reduces the inward rectification of the current. Flecainide interacts with the HI loop of the cytoplasmic domain of the channel, Cys311 being critical for the effect. This explains why flecainide does not increase I(Kir2.2) and I(Kir2.3), because Kir2.2 and Kir2.3 channels do not exhibit a Cys residue at the equivalent position. We further show that incubation with flecainide increases expression of functional Kir2.1 channels in the membrane, an effect also determined by Cys311. Indeed, flecainide pharmacologically rescues R67W, but not R218W, channel mutations found in Andersen syndrome patients. Moreover, our findings provide noteworthy clues about the structural determinants of the C terminus cytoplasmic domain of Kir2.1 channels involved in the control of gating and rectification.

Specific residues of the cytoplasmic domains of cardiac inward rectifier potassium channels are effective antifibrillatory targets.

Atrial and ventricular tachyarrhythmias can be perpetuated by up-regulation of inward rectifier potassium channels. Thus, it may be beneficial to block inward rectifier channels under conditions in which their function becomes arrhythmogenic (e.g., inherited gain-of-function mutation channelopathies, ischemia, and chronic and vagally mediated atrial fibrillation). We hypothesize that the antimalarial quinoline chloroquine exerts potent antiarrhythmic effects by interacting with the cytoplasmic domains of Kir2.1 (I(K1)), Kir3.1 (I(KACh)), or Kir6.2 (I(KATP)) and reducing inward rectifier potassium currents. In isolated hearts of three different mammalian species, intracoronary chloroquine perfusion reduced fibrillatory frequency (atrial or ventricular), and effectively terminated the arrhythmia with resumption of sinus rhythm. In patch-clamp experiments chloroquine blocked I(K1), I(KACh), and I(KATP). Comparative molecular modeling and ligand docking of chloroquine in the intracellular domains of Kir2.1, Kir3.1, and Kir6.2 suggested that chloroquine blocks or reduces potassium flow by interacting with negatively charged amino acids facing the ion permeation vestibule of the channel in question. These results open a novel path toward discovering antiarrhythmic pharmacophores that target specific residues of the cytoplasmic domain of inward rectifier potassium channels.

Thiopental inhibits function of different inward rectifying potassium channel isoforms by a similar mechanism.

Thiopental is a well-known intravenous barbiturate anesthetic with important cardiac side effects. The actions of thiopental on the transmembrane ionic currents that determine the resting potential and action potential duration in cardiomyocytes have been studied widely. We aimed at elucidating the characteristics and mechanism of inhibition by thiopental on members of the subfamily of inward rectifying Kir2.x (Kir2.1, 2.2 and 2.3), Kir1.1 and Kir6.2/SUR2A channels. These inward rectifier potassium channels were transfected in HEK-293 cells and macroscopic currents were recorded in the whole-cell and inside-out configurations of the patch-clamp technique. Thiopental inhibited Kir2.1, Kir2.2, Kir2.3, Kir1.1 and Kir6.2/SUR2A currents with similar potency; in whole-cell experiments 30 microM thiopental decreased Kir2.1, Kir2.2, Kir2.3 and Kir1.1 currents to 55+/-6, 39+/-8, 42+/-5 and 49+/-5% at -120 mV, respectively. Point mutations on Kir2.3 (I213L) or Kir2.1 (L222I) did not modify the potency of block. Thiopental inhibited all Kir channels in a concentration-dependent and voltage-independent manner. Also, the time course of thiopental inhibition was slow (T(1/2) approximately 4 min) and independent of external or internal drug application. However, in the presence of PIP(2), inhibition by thiopental on Kir2.1 was significantly decreased. Thiopental at clinically relevant concentrations significantly inhibited all Kir channels evaluated in this work. The reduction of thiopental effects during PIP(2) treatment suggests that thiopental inhibition on Kir2.1 channels is related to channel-PIP(2) interaction.

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.

Tamoxifen inhibits inward rectifier K+ 2.x family of inward rectifier channels by interfering with phosphatidylinositol 4,5-bisphosphate-channel interactions.

Tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibits the inward rectifier potassium current (I(K1)) in cardiac myocytes by an unknown mechanism. We characterized the inhibitory effects of tamoxifen on Kir2.1, Kir2.2, and Kir2.3 potassium channels that underlie cardiac I(K1). We also studied the effects of 4-hydroxytamoxifen and raloxifene. All three drugs inhibited inward rectifier K(+) 2.x (Kir2.x) family members. The order of inhibition for all three drugs was Kir2.3 > Kir2.1 approximately Kir2.2. The onset of inhibition of Kir2.x current by these compounds was slow (T(1/2) approximately 6 min) and only partially recovered after washout ( approximately 30%). Kir2.x inhibition was concentration-dependent but voltage-independent. The time course and degree of inhibition was independent of external or internal drug application. We tested the hypothesis that tamoxifen interferes with the interaction between the channel and the membrane-delimited channel activator, phosphatidylinositol 4,5-bisphosphate (PIP(2)). Inhibition of Kir2.3 currents was significantly reduced by a single point mutation of I213L, which enhances Kir2.3 interaction with membrane PIP(2). Pretreatment with PIP(2) significantly decreased the inhibition induced by tamoxifen, 4-hydroxytamoxifen, and raloxifene on Kir2.3 channels. Pretreatment with spermine (100 microM) decreased the inhibitory effect of tamoxifen on Kir2.1, probably by strengthening the channel's interaction with PIP(2). In cat atrial and ventricular myocytes, 3 microM tamoxifen inhibited I(K1), but the effect was greater in the former than the latter. The data strongly suggest that tamoxifen, its metabolite, and the estrogen receptor inhibitor raloxifene inhibit Kir2.x channels indirectly by interfering with the interaction between the channel and PIP(2).