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.T he cardiac inwardly rectifying K + current (I K1 ) stabilizes resting membrane potential (RMP) close to the reversal potential of K + (E K ) and shapes the final repolarization phase of the action potential (AP) (1). Three inwardly rectifying channels (Kir2.1, Kir2.2, and Kir2.3) contribute to I K1 in the human heart assembled as homo-and/or heterotetramers (2). Experimental data suggest that in humans, Kir2.1 is the major isoform underlying ventricular I K1 , whereas its relative contribution to atrial I K1 seems to be lower (3). The strong inward rectification of Kir2.x channels, i.e., the preferential conduction of inward compared with outward current, depends on the binding of intracellular Mg 2+ and polyamines to the cytoplasmic pore and to the inner vestibule of the channel (4).Gain-and loss-of-function mutations in the gene that encodes Kir2.1 (KCNJ2) have been reported, and both the I K1 increase and decrease produced by these mutations are associated with severe ventricular arrhythmias (1). Furthermore, experimental data showed that as the amplitude of the outward component of the I K1 increases, the frequency of the fast and stable reentry of spiral waves (rotors) increases. Indeed, the importance of I K1 in the establishment of rotors and ventricular fibrillation dynamics has been shown (5).Flecainide is a class I antiarrhythmic drug that, besides its Na + channel-blocking properties, exhibits class III antiarrhythmic effects [i.e., prolongs AP duration (APD) and refractoriness] at the atrial but not at the ventricular le...

Background Genetic defects in KCNJ8, encoding the Kir6.1 subunit of the ATP-sensitive K+ channel (IK-ATP), have previously been associated with early repolarization (ERS) and Brugada (BrS) syndromes. Here we test the hypothesis 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 −18.0mV (p<0.01) and increased late INa from 0.14% to 2.01% of peak INa (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 IK-ATP when coupled with a loss-of-function in SCN5A may underlie type 3 ERS, which is associated with a severe arrhythmic phenotype.

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.

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 ϩ 2.x (Kir2.x) family members. The order of inhibition for all three drugs was Kir2.3 Ͼ Kir2.1 ϳ Kir2.2. The onset of inhibition of Kir2.x current by these compounds was slow (T 1/2 ϳ 6 min) and only partially recovered after washout (ϳ30%). 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 M) 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 M 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 .

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.

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.

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 μM 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.

-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-{␤[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 Ͼ10-fold over the traditional patchclamp 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. optical action potential; preclinical drug screening; QT prolongation; acquired long QT syndrome; stem cell model RECENT ADVANCES in induced pluripotent stem cell (iPSC) technology now allow for the routine derivation of patient-and disease-specific human iPSC cardiomyocyte (iPSC-CM) models of cardiovascular disease. The impact of this technology has far-reaching implications, ranging from drug discovery, preclinical drug screening, mechanistic understanding of disease processes, and approaches for personalized medicine. Human iPSC-CM-based assays are emerging as a promising tool for the in vitro preclinical screening of QT intervalprolonging 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. The patch-clamp technique allows for direct measurement of transmembrane voltage (V m ) to precisely quantify the morphology and duration of atrial and ventricular action potentials (APs). However, the patch-clamp technique is a labor-intensive technique, which involves highly skilled, experienced individuals. Recently, the utility of a genetically encoded voltage-sensitive fluorescent reporter (ArcLight) was described in human embryonic stem cell-derived CMs (6). This reporter generated robust fluorescent signals from single cells, allowing for moderate-high throughput analyses, although the temporal response was delayed relative to directly measured V m . While voltage-sensitive dyes such as di-4-ANNEPS and di-8-ANNEPS allow ...