Inhibition of the Inositol Kinase Itpkb Augments Calcium Signaling in Lymphocytes and Reveals a Novel Strategy to Treat Autoimmune Disease.

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca2+ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease.

Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation.

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.

The development of automated patch clamp assays for canonical transient receptor potential channels TRPC3, 6, and 7.

The canonical transient receptor potential channel subfamily (TRPC3, TRPC6, and TRPC7) contains Ca(2+) permeable non-selective cation channels that are widely expressed in a variety of tissues. There is increasing evidence implicating TRPC channels, particularly TRPC3 and 6, in physiological and pathophysiological processes, eliciting interest in these channels as novel drug targets. Electrophysiology remains a benchmark technique for measuring ion channel function and accurately determining the pharmacological effects of compounds. In this report we describe the development of TRPC inhibitor assays on 2 automated planar patch clamp platforms-the IonWorks(®) Quattro™ and QPatch(®) systems. To enable activation of TRPC channels by carbachol, Chinese Hamster Ovary-K1 cells stably expressing the muscarinic M3 receptor were transduced with human TRPC3, TRPC6, or TRPC7 using BacMam viruses. TRPC3, 6, and 7 currents could be recorded on both platforms. However, the design of each platform limits which assay parameters can be recorded. Due to its continuous recording capabilities, the QPatch can capture both the activation and decay of the response. However, the transient nature of TRPC channels, the inability to reactivate and the large variation in peak currents limits the ability to develop assays for compound screening. The IonWorks Quattro, due to its discontinuous sampling, did not fully capture the peak of TRPC currents. However, due to the ability of the IonWorks Quattro to record from 64 cells per well, the variation from well to well was sufficiently reduced allowing for the development of medium-throughput screening assays.

Increased vulnerability of human ventricle to re-entrant excitation in hERG-linked variant 1 short QT syndrome.

The short QT syndrome (SQTS) is a genetically heterogeneous condition characterized by abbreviated QT intervals and an increased susceptibility to arrhythmia and sudden death. This simulation study identifies arrhythmogenic mechanisms in the rapid-delayed rectifier K(+) current (I(Kr))-linked SQT1 variant of the SQTS. Markov chain (MC) models were found to be superior to Hodgkin-Huxley (HH) models in reproducing experimental data regarding effects of the N588K mutation on KCNH2-encoded hERG. These ionic channel models were then incorporated into human ventricular action potential (AP) models and into 1D and 2D idealised and realistic transmural ventricular tissue simulations and into a 3D anatomical model. In single cell models, the N588K mutation abbreviated ventricular cell AP duration at 90% repolarization (APD(90)) and decreased the maximal transmural voltage heterogeneity (δV) during APs. This resulted in decreased transmural heterogeneity of APD(90) and of the effective refractory period (ERP): effects that are anticipated to be anti-arrhythmic rather than pro-arrhythmic. However, with consideration of transmural heterogeneity of I(Kr) density in the intact tissue model based on the ten Tusscher-Noble-Noble-Panfilov ventricular model, not only did the N588K mutation lead to QT-shortening and increases in T-wave amplitude, but δV was found to be augmented in some local regions of ventricle tissue, resulting in increased tissue vulnerability for uni-directional conduction block and predisposing to formation of re-entrant excitation waves. In 2D and 3D tissue models, the N588K mutation facilitated and maintained re-entrant excitation waves due to the reduced substrate size necessary for sustaining re-entry. Thus, in SQT1 the N588K-hERG mutation facilitates initiation and maintenance of ventricular re-entry, increasing the lifespan of re-entrant spiral waves and the stability of scroll waves in 3D tissue.

Acidosis impairs the protective role of hERG K(+) channels against premature stimulation.

INTRODUCTION: Potassium channels encoded by human ether-à-go-go-related gene (hERG) underlie the cardiac rapid delayed rectifier K(+) channel current (I(Kr)). Acidosis occurs in a number of pathological situations and modulates a range of ionic currents including I(Kr) . The aim of this study was to characterize effects of extracellular acidosis on hERG current (I(hERG)), with particular reference to quantifying effects on I(hERG) elicited by physiological waveforms and upon the protective role afforded by hERG against premature depolarizing stimuli. METHODS AND RESULTS: I(hERG) recordings were made from hERG-expressing Chinese Hamster Ovary cells using whole-cell patch-clamp at 37°C. I(hERG) during action potential (AP) waveforms was rapidly suppressed by reducing external pH from 7.4 to 6.3. Peak repolarizing current and steady state I(hERG) activation were shifted by ∼+6 mV; maximal I(hERG) conductance was reduced. The voltage-dependence of I(hERG) inactivation was little-altered. Fast and slow time-constants of I(hERG) deactivation were smaller across a range of voltages at pH 6.3 than at pH 7.4, and the contribution of fast deactivation increased. A modest acceleration of the time-course of recovery of I(hERG) from inactivation was observed, but time-course of activation was unaffected. The amplitude of outward I(hERG) transients elicited by premature stimuli following an AP command was significantly decreased at lower pH. Computer simulations showed that after AP repolarization a subthreshold stimulus at pH 7.4 could evoke an AP at pH 6.3. CONCLUSION: During acidosis the contribution of I(hERG) to action potential repolarization is reduced and hERG may be less effective in counteracting proarrhythmogenic depolarizing stimuli.

Action potential clamp and chloroquine sensitivity of mutant Kir2.1 channels responsible for variant 3 short QT syndrome.

Recently identified genetic forms of short QT syndrome (SQTS) are associated with an increased risk of arrhythmia and sudden death. The SQT3 variant is associated with an amino-acid substitution (D172N) in the KCNJ2-encoded Kir2.1 K+ channel. In this study, whole-cell action potential (AP) clamp recording from transiently transfected Chinese Hamster Ovary cells at 37 degrees C showed marked augmentation of outward Kir2.1 current through D172N channels, associated with right-ward voltage-shifts of peak repolarizing current during both ventricular and atrial AP commands. Peak outward current elicited by ventricular AP commands was inhibited by chloroquine with an IC50 of 2.45 microM for wild-type (WT) Kir2.1, of 3.30 microM for D172N-Kir2.1 alone and of 3.11 microM for co-expressed WT and D172N (P>0.05 for all). These findings establish chloroquine as an effective inhibitor of SQT3 mutant Kir2.1 channels.

The hERG potassium channel and hERG screening for drug-induced torsades de pointes.

Drug-induced torsades de pointes (TdP) arrhythmia is a major safety concern in the process of drug design and development. The incidence of TdP tends to be low, so early pre-clinical screens rely on surrogate markers of TdP to highlight potential problems with new drugs. hERG (human ether-à-go-go-related gene, alternative nomenclature KCNH2) is responsible for channels mediating the 'rapid' delayed rectifier K+ current (IKr) which plays an important role in ventricular repolarization. Pharmacological inhibition of native IKr and of recombinant hERG channels is a shared feature of diverse drugs associated with TdP. In vitro hERG assays therefore form a key element of an integrated assessment of TdP liability, with patch-clamp electrophysiology offering a 'gold standard'. However, whilst clearly necessary, hERG assays cannot be assumed automatically to provide sufficient information, when considered in isolation, to differentiate 'safe' from 'dangerous' drugs. Other relevant factors include therapeutic plasma concentration, drug metabolism and active metabolites, severity of target condition and drug effects on other cardiac ion channels that may mitigate or exacerbate effects of hERG blockade. Increased understanding of the nature of drug-hERG channel interactions may ultimately help eliminate potential hERG blockade early in the design and development process. Currently, for promising drug candidates integration of data from hERG assays with information from other pre-clinical safety screens remains essential.

Disopyramide is an effective inhibitor of mutant HERG K+ channels involved in variant 1 short QT syndrome.

The recently identified idiopathic short QT syndrome (SQTS) is associated with an increased risk of arrhythmia and sudden death. The use of implantable cardioverter defibrillators helps to protect SQTS patients from ventricular fibrillation; however, pharmacological treatments to normalise the QT interval are limited: thus far only quinidine has been found to be effective in a subset of patients, with the SQT1 variant. SQT1 is associated with an amino acid substitution (N588K) in the KCNH2-encoded HERG K(+) channel that reduces HERG current (I(HERG)) inactivation and sensitivity to drug block. We demonstrate here that the N588K-HERG mutation only slightly attenuates I(HERG) blockade by the Class Ia antiarrhythmic drug disopyramide (1.5-fold elevation of IC(50)), compared to quinidine (3.5-fold elevation of IC(50)) and the Class III antiarrhythmic drug E-4031 (11.5-fold elevation of IC(50)). Thus, of the drugs studied to date, disopyramide is the one least affected by the SQT1 HERG mutation. Disopyramide is associated with QT prolongation in normal use and our findings provide a rational basis for its evaluation as a treatment for SQT1.

Inhibition of the HERG K+ channel by the antifungal drug ketoconazole depends on channel gating and involves the S6 residue F656.

The mechanism of human ether-à-go-go-related gene (HERG) K+ channel blockade by the antifungal agent ketoconazole was investigated using patch-clamp recording from mammalian cell lines. Ketoconazole inhibited whole-cell HERG current (IHERG) with a clinically relevant half-maximal inhibitory drug concentration (IC50) value of 1.7 microM. The voltage- and time-dependent characteristics of IHERG blockade by ketoconazole indicated dependence of block on channel gating, ruling out a significant role for closed-state channel inhibition. The S6 HERG mutations Y652A and F656A produced approximately 4-fold and approximately 21-fold increases in IC50 for IHERG blockade, respectively. Thus, ketoconazole accesses the HERG channel pore-cavity on channel gating, and the S6 residue F656 is an important determinant of ketoconazole binding.

Short QT syndrome.

The idiopathic short QT syndrome (SQTS) is a recently identified condition characterized by abbreviated QT intervals (typically 300 ms or less) and in affected families is associated with an increased incidence of atrial and ventricular arrhythmias and sudden cardiac death. Genetic analysis has, to date, identified three distinct forms of the condition, involving gain-of-function mutations to three different cardiac potassium channel genes: KCNH2 (SQT1), KCNQ1 (SQT2) and KCNJ2 (SQT3). This article reviews recent advances in understanding this syndrome, discussing the basis of QT interval shortening, possible mechanisms for the associated arrhythmogenic risk in SQT1, current approaches to treatment of the SQTS (focusing on SQT1) and avenues for future investigation.

The N588K-HERG K+ channel mutation in the 'short QT syndrome': mechanism of gain-in-function determined at 37 degrees C.

The idiopathic short QT syndrome (SQTS) is characterised by an abnormally short QT interval on the electrocardiogram and by an increased risk of arrhythmia and sudden death. One variant of the syndrome is linked to missense mutations that lead to a single amino-acid change (N588K; asparagine to lysine) in the S5-Pore linker region of the cardiac HERG K(+) channel. This study was performed in order to determine how the N588K mutation alters HERG channel current (I(HERG)) kinetics at mammalian physiological temperature. The whole-cell current-voltage (I-V) relation for wild-type (WT) I(HERG) measured from Chinese Hamster Ovary cells was maximal at approximately 0 mV and showed marked inward rectification positive to this. In contrast, N588K I(HERG) showed marked rectification only at +60 mV and at more positive voltages. The voltage dependence of activation of N588K-HERG did not differ significantly from that of WT-HERG. However, N588K I(HERG) had a significantly more positive inactivation V(0.5) (-8.14+/-0.82 mV) than did WT I(HERG) (-70.05+/-0.82 mV; P<0.001, unpaired t test; n=5 for each). Its P(Na)/P(K) ratio was also greater. The instantaneous I-V relation for N588K I(HERG) under action potential voltage clamp peaked at approximately +40 mV, compared to approximately -37 mV for WT-I(HERG). These findings underscore the importance of the S5-P linker in HERG channel function and indicate that N588K-HERG contributes increased repolarising current earlier in the ventricular action potential at physiological temperature due to a approximately +60 mV shift in voltage dependence of I(HERG) inactivation.

Role of catalase in the smooth muscle relaxant actions of sodium azide and cyanamide.

The aim of this study was to determine the role of catalase in the smooth muscle relaxant actions of sodium azide and cyanamide. The effects of 3-amino-1,2,4-triazole suggested a role for this enzyme in the relaxant actions of sodium azide on rat aorta and bovine retractor penis muscle and cyanamide on rat aorta. Moreover, results obtained using a difference spectrophotometric assay based upon the oxidation of haemoglobin were consistent with the catalase-dependent oxidation of sodium azide to nitric oxide (NO) and of cyanamide to nitroxyl anion. Surprisingly, however, no free nitric oxide or nitroxyl was detected in solution using a sensitive electrode. This anomaly might be explained if the stable complexes of catalase with nitric oxide or nitroxyl do not release their respective ligand except to sites of high affinity, such as the haemoglobin employed in the difference spectrophotometric assay, or indeed, the soluble guanylate cyclase within the smooth muscle.