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Background and Purpose: The canonical Kir6.2/SUR2A ventricular KATP channel is highly ATP‐sensitive and remains closed under normal physiological conditions. These channels activate only when prolonged metabolic compromise causes significant ATP depletion and then shortens the action potential to reduce contractile activity. Pharmacological activation of KATP channels is cardioprotective, but physiologically, it is difficult to understand how these channels protect the heart if they only open under extreme metabolic stress. The presence of a second KATP channel population could help explain this. Here, we characterise the biophysical and pharmacological behaviours of a constitutively active Kir6.1‐containing KATP channel in ventricular cardiomyocytes. Experimental Approach: Patch‐clamp recordings from rat ventricular myocytes in combination with well‐defined pharmacological modulators was used to characterise these newly identified K+ channels. Action potential recording, calcium (Fluo‐4) fluorescence measurements and video edge detection of contractile function were used to assess functional consequences of channel modulation. Key Results: Our data show a ventricular K+ conductance whose biophysical characteristics and response to pharmacological modulation were consistent with Kir6.1‐containing channels. These Kir6.1‐containing channels lack the ATP‐sensitivity of the canonical channels and are constitutively active. Conclusion and Implications: We conclude there are two functionally distinct populations of ventricular KATP channels: constitutively active Kir6.1‐containing channels that play an important role in fine‐tuning the action potential and Kir6.2/SUR2A channels that activate with prolonged ischaemia to impart late‐stage protection against catastrophic ATP depletion. Further research is required to determine whether Kir6.1 is an overlooked target in Comprehensive in vitro Proarrhythmia Assay (CiPA) cardiac safety screens. [ABSTRACT FROM AUTHOR]

Introduction: Stochastic trajectory analysis of ranked signals (STAR) is a novel method for mapping arrhythmia. The aim was to describe its development and validation as a mapping tool. Methods and Results: The method ranks electrodes in terms of the proportion of the time they lead relative to neighboring electrodes and ascribes a predominant direction of activation between electrodes. This was conceived with the aim of mapping atrial fibrillation (AF) drivers. Validation of this approach was performed in stages. First, in vitro simultaneous multi‐electrode array and optical mapping were performed on spontaneously fibrillating HL1 cell cultures, to determine if such a method would be able to determine early sites of activation (ESA). A clinical study acquiring unipolar electrograms using a 64‐pole basket for the purposes of STAR mapping in patients undergoing atrial tachycardia (AT) ablation. STAR maps were analyzed by physicians to see if arrhythmia mechanisms could be correctly determined. Mapping was then repeated during atrial pacing. STAR mapping of in vitro activation sequences accurately correlated to the optical maps of planar and rotational activation. Thirty‐two ATs were mapped in 25 patients. The ESA accurately identified focal/micro‐reentrant ATs and the mechanism of macro‐reentrant ATs was effectively demonstrated. STAR method accurately identified four pacing sites in all patients. Conclusions: This novel STAR method correlated well with the gold standard of optical mapping in vitro and was able to accurately identify AT mechanisms. Further analysis is needed to determine whether the method might be of use mapping AF. [ABSTRACT FROM AUTHOR]

ATP-sensitive potassium channels ( KATP) are widely distributed and present in a number of tissues including muscle, pancreatic beta cells and the brain. Their activity is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels. Thus, they link cellular metabolism with membrane excitability. Recent studies using genetically modified mice and genomic studies in patients have implicated KATP channels in a number of physiological and pathological processes. In this review, we focus on their role in cellular function and protection particularly in the cardiovascular system. [ABSTRACT FROM AUTHOR]

Background and Purpose: Selective and potent antagonists for the β(2) -adrenoceptor are potentially interesting as experimental and clinical tools, and we sought to identify novel ligands with this pharmacology.Experimental Approach: A range of pharmacological assays was used to assess potency, affinity, selectivity (β(2) -adrenoceptor vs. β(1) -adrenoceptor) and efficacy.Key Results: Ten novel compounds were identified but none had as high affinity as the prototypical β(2) -adrenoceptor blocker ICI-118,551, although one of the novel compounds was more selective for β(2) -adrenoceptors. Most of the ligands were inverse agonists for β(2) -adrenoceptor-cAMP signalling, although one (5217377) was a partial agonist and another a neutral antagonist (7929193). None of the ligands were efficacious with regard to β(2) -adrenoceptor-β-arrestin signalling. The (2S,3S) enantiomers were identified as the most active, although unusually the racemates were the most selective for the β(2) -adrenoceptors. This was taken as evidence for some unusual enantiospecific behaviour.Conclusions and Implications: In terms of improving on the pharmacology of the ligand ICI-118,551, one of the compounds was more selective (racemic JB-175), while one was a neutral antagonist (7929193), although none had as high an affinity. The results substantiate the notion that β-blockers do more than simply inhibit receptor activation, and differences between the ligands could provide useful tools to investigate receptor biology. [ABSTRACT FROM AUTHOR]

Background and Purpose: A class of drugs known as K(ATP) -channel openers induce cardioprotection. This study examined the effects of the novel K(ATP) -channel opener, the fluorine-containing pinacidil derivative, flocalin, on cardiac-specific K(ATP) -channels, excitability of native cardiac myocytes and on the ischaemic heart.Experimental Approach: The action of flocalin was investigated on: (i) membrane currents through cardiac-specific K(ATP) -channels (I(KATP) ) formed by K(IR) 6.2/SUR2A heterologously expressed in HEK-293 cells (HEK-293(₆.₂/₂A) ); (ii) excitability and intracellular Ca²(+) ([Ca²(+) ](i) ) transients of cultured rat neonatal cardiac myocytes; and (iii) functional and ultrastructural characteristics of isolated guinea-pig hearts subjected to ischaemia-reperfusion.Key Results: Flocalin concentration-dependently activated a glibenclamide-sensitive I(KATP) in HEK-293(₆.₂/₂A) cells with an EC₅₀= 8.1 ± 0.4 µM. In cardiac myocytes, flocalin (5 µM) hyperpolarized resting potential by 3-5 mV, markedly shortened action potential duration, reduced the amplitude of [Ca²(+) ](i) transients by 2-3-fold and suppressed contraction. The magnitude and extent of reversibility of these effects depended on the type of cardiac myocytes. In isolated hearts, perfusion with 5 µmol·L⁻¹ flocalin, before inducing ischaemia, facilitated restoration of contraction during reperfusion, decreased the number of extrasystoles, prevented the appearance of coronary vasoconstriction and reduced damage to the cardiac tissue at the ultrastructural level (state of myofibrils, membrane integrity, mitochondrial cristae structure).Conclusion and Implications: Flocalin induced potent cardioprotection by activating cardiac-type K(ATP) -channels with all the benefits of the presence of fluorine group in the drug structure: higher lipophilicity, decreased toxicity, resistance to oxidation and thermal degradation, decreased metabolism in the organism and prolonged therapeutic action. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is a key component in signal transduction, being a precursor to other signalling molecules and itself associated with roles in signal transduction and cell biology. Tubby is a membrane bound transcription factor whose dysfunction results in obesity in mice. It contains a domain that selectively binds PtdIns(4,5)P2. We have investigated the use of a fluorescently tagged version of this domain to monitor changes in PtdIns(4,5)P2 concentration in living cells and compared it to the pleckstrin homology domain of PLCδ1. Our results show that selected mutants of this domain report receptor-mediated changes in cellular PtdIns(4,5)P2. In contrast to the pleckstrin homology domain of PLCδ1 it does not have a significant affinity for inositol 1,4,5-trisphosphate (IP3). Using a selected mutant, we examine the regulation of ATP-sensitive K+ channels via a Gq/11-coupled receptor. These experiments reveal a correlation between reporter translocation and the onset of current inhibition whilst the recovery of current after agonist removal is delayed when compared to the reporter. Furthermore our studies reveal the importance of Ca2+ in determining the overall activity of phospholipase C in living cells. This probe may be valuable in examining changes in PtdIns(4,5)P2 distinct from those of IP3 in intact cells in a variety of physiological settings. [ABSTRACT FROM AUTHOR]

M-channels are voltage-gated K+ channels that regulate the excitability of many neurons. They are composed of Kv7 (KCNQ) family subunits, usually Kv7.2 + Kv7.3. Native M-channels and expressed Kv7.2 + 7.3 channels are inhibited by stimulating Gq/11-coupled receptors – prototypically the M1 muscarinic acetylcholine receptor (M1-mAChR). The channels require membrane phosphatidylinositol-4,5-bisphosphate (PIP2) to open and the effects of mAChR stimulation result primarily from the reduction in membrane PIP2 levels following Gq/phospholipase C-catalysed PIP2 hydrolysis. However, in sympathetic neurons, M-current inhibition by bradykinin appears to be mediated through the release and action of intracellular Ca2+ by inositol-1,4,5-trisphosphate (IP3), a product of PIP2 hydrolysis, rather than by PIP2 depletion. We have therefore compared the effects of bradykinin and oxotremorine-M (a muscarinic agonist) on membrane PIP2 in sympathetic neurons using a fluorescently tagged mutated C-domain of the PIP2 binding probe, ‘tubby’. In concentrations producing equal M-current inhibition, bradykinin produced about one-quarter of the reduction in PIP2 produced by oxotremorine-M, but equal reduction when PIP2 synthesis was blocked with wortmannin. Likewise, wortmannin restored bradykinin-induced M-current inhibition when Ca2+ release was prevented with thapsigargin. Thus, inhibition by bradykinin can use product (IP3/Ca2+)-dependent or substrate (PIP2) dependent mechanisms, depending on Ca2+ availability and PIP2 synthesis rates. [ABSTRACT FROM AUTHOR]

Objective Congenital hyperinsulinism (CHI) is a cause of persistent and severe hypoglycaemia in infancy. Mutations in the genes ABCC8 and KCNJ11 encoding SUR1 and Kir6·2, respectively, are the commonest cause of CHI. We investigated whether the possession of two DNA variants leading to coding changes in a single allele of ABCC8 can affect the potential mechanism of disease pathogenesis. Design and patients We studied two patients with complex mutations in the ABCC8 gene with CHI and used in vitro studies to explore the potential disease mechanism and the contribution of the various mutant allelles. Results The first case had diffuse disease and was homozygous for the mutations D1193V and R1436Q in SUR1. Channel complexes containing the D1193V mutant were delivered to the plasma membrane and were functional and those containing R1436Q were also present at the plasma membrane but were nonfunctional. Combining the two mutations (SUR1D1193V/R1436Q) led to intracellular retention of the channel complex. In a second family, the patient had histologically focal disease and was heterozygous for two mutations from his father (G228D and D1471N) and one from his mother (V1572I). SUR1 G228D and D1471N singly or in combination led to intracellular retention of the channel complex and loss of function. By contrast, V1572I is trafficked appropriately and is functional, consistent with a mechanism of reduction to hemizygosity of paternal ABCC8 in focal disease. V1572I is likely to be a benign DNA variant. Conclusion In one patient the combination of two coding variants led to intracellular retention of channel complex. In a second patient, functional studies allowed us to unravel the DNA variants likely to be causing the abrogation of ATP-sensitive K+ channel function. [ABSTRACT FROM AUTHOR]

1. We investigated the mechanism of action of two novel nonsulphonylurea ATP-sensitive potassium channel (K(ATP)) inhibitors, PNU-99963 and PNU-37883A, on four types of cloned K(ATP) channels. 2. Whole-cell currents were recorded in a symmetrical potassium (140 mM) gradient in HEK-293 cells stably expressing Kir6.2/SUR1, Kir6.2/SUR2A, Kir6.2/SUR2B or Kir6.1/SUR2B. 3. PNU-99963 potently inhibited the four K(ATP) channel clones. The concentration at which half-maximum current was inhibited (IC(50)) was 66, 41, 43 and 11 nM for Kir6.2/SUR1, Kir6.2/SUR2A, Kir6.2/SUR2B and Kir6.1/SUR2B, respectively. In contrast, PNU-99963 up to a concentration of 3 microM had no significant effect on current generated in HEK-293 cells by transiently expressing Kir6.2Delta26, a C-terminal truncated pore-forming subunit of Kir6.2. 4. PNU-37883A inhibited four types of K(ATP) channels, but to different extents. Inhibition of the putative smooth muscle K(ATP) channel types, Kir6.2/SUR2B (IC(50); 15 microM) and Kir6.1/SUR2B (IC(50); 6 microM), was significantly greater than inhibition of either the pancreatic beta cell or cardiac K(ATP) channel clones. Moreover, PNU-37883A significantly inhibited currents generated by expressing Kir6.2Delta26 alone, with an IC(50) of 5 microM, which was significantly increased to 38 microM when Kir6.2Delta26 was expressed with SUR2B. 5. In conclusion, two structurally different nonsulphonylurea compounds, PNU-99963 and PNU-37883A, inhibit K(ATP) channels via different mechanisms, namely through the sulphonylurea receptor (SUR) and the pore-forming subunits, respectively, although SUR2B reduced the inhibitory effect of PNU-37883A. While PNU-99963 potently inhibits all the four cloned K(ATP) channels, PNU-37883A has a degree of selectivity towards both smooth muscle K(ATP) channels, but could not discriminate between them. [ABSTRACT FROM AUTHOR]

1 We investigated the mechanism of action of two novel nonsulphonylurea ATP-sensitive potassium channel (K[SUBATP]) inhibitors, PNU-99963 and PNU-37883A, on four types of cloned K[SUBATP] channels. 2 Whole-cell currents were recorded in a symmetrical potassium (140 mM) gradient in HEK-293 cells stably expressing Kir6.2/SUR1, Kr6.2/SUR2A. Kr6.2/SUR2B or Kir6.1/SUP2B. 3 PNU-99963 potently inhibited the four K[SUBATP] channel clones. The concentration at which half-maximum current was inhibited (IC[SUB50]) was 66, 41, 43 and 11 nM for Kir6.2/SUR1, KR6.2/SUR2A, Kir6.2/SUR2B and Kir6.1/SUR2B, respectively. In contrast, PNU-99963 up to a concentration of 3μM had no significant effect on current generated in HEK-293 cells by transiently expressing Kir6.2Δ26, a C-terminal truncated pore-forming subunit of Kir6.2 4 PNU-37883A inhibited four types of K[SUBATP] channels, but to different extents. Inhibition of the putative smooth muscle K[SUBATP] channel types. Kir6.2/SUR2B (IC[SUB50]; 15 μM) and Kir6.1/SUR2B (IC[SUB50]; 6 μM), was significantly greater than inhibition of either the pancreatic β cell or cardiac K[SUBATP] channel clones. Moreover, PNU-37883A significantly inhibited currents generated by expressing Kir6.2Δ26 alone, with an IC[SUB50] of 5 μM, which was significantly increased to 38 μM when Kir6.2Δ26 was expressed with SUR2B. 5 In conclusion, two structurally different nonsulphonylurea compounds, PNU-99963 and PNU-37883A, inhibit K[SUBATP] channels via different mechanisms, namely through the sulphonylurea receptor (SUR), and the pore-forming subunits, respectively, although SUR2B reduced the inhibitory effects of PNU-37883A. While PNU-99963 potently inhibits all the four cloned K[SUBATP] channels, PNU-37883A has a degree of selectively towards both smooth muscle K[SUBATP] channels, but could not discriminate between them. [ABSTRACT FROM AUTHOR]

ATP-sensitive K+ channels are an octameric assembly of two proteins, a sulfonylurea receptor (SUR1) and an ion conducting subunit (Kir 6.0). We have examined the role of the C-terminus of SUR1 by expressing a series of truncation mutants together with Kir6.2 stably in HEK293 cells. Biochemical analyses using coimmunoprecipitation indicate that SUR1 deletion mutants and Kir6.2 assemble and that a SUR1 deletion mutant binds glibenclamide with high affinity. Electrophysiological recordings indicate that ATP sensitivity is normal but the response of the mutant channel complexes to tolbutamide, MgADP and diazoxide is disturbed. Quantitative immunofluorescence and cell surface biotinylation supports the idea that there is little disturbance in the efficiency of trafficking. Our data show that deletions of the C-terminal most cytoplasmic domain of SUR1, can result in functional channels at the plasma membrane in mammalian cells that have an abnormal response to physiological and pharmacological agents. [ABSTRACT FROM AUTHOR]

Background: Ventricular repolarization time (ECG QT and JT intervals) is associated with malignant arrhythmia. Genome-wide association studies have identified 230 independent loci for QT and JT; however, 50% of their heritability remains unexplained. Previous work supports a causal effect of lower serum calcium concentrations on longer ventricular repolarization time. We hypothesized calcium interactions with QT and JT variant associations could explain a proportion of the missing heritability., Methods and Results: We performed genome-wide calcium interaction analyses for QT and JT intervals. Participants were stratified by their calcium level relative to the study distribution (top or bottom 20%). We performed a 2-stage analysis (genome-wide discovery [N=62 532] and replication [N=59 861] of lead variants) and a single-stage genome-wide meta-analysis (N=122 393, [European ancestry N=117 581, African ancestry N=4812]). We also calculated 2-degrees of freedom joint main and interaction and 1-degree of freedom interaction P values. In 2-stage and single-stage analyses, 50 and 98 independent loci, respectively, were associated with either QT or JT intervals (2-degrees of freedom joint main and interaction P value <5×10 -8 ). No lead variant had a significant interaction result after correcting for multiple testing and sensitivity analyses provided similar findings. Two loci in the single-stage meta-analysis were not reported previously ( SPPL2B and RFX6 )., Conclusions: We have found limited support for an interaction effect of serum calcium on QT and JT variant associations despite sample sizes with suitable power to detect relevant effects. Therefore, such effects are unlikely to explain a meaningful proportion of the heritability of QT and JT, and factors including rare variation and other environmental interactions need to be considered.