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The electrocardiogram (ECG) is a commonly used clinical tool that reflects cardiac excitability and disease. Many parameters are can be measured and with the improvement of methodology can now be quantified in an automated fashion, with accuracy and at scale. Furthermore, these measurements can be heritable and thus genome wide association studies inform the underpinning biological mechanisms. In this review we describe how we have used the resources in UK Biobank to undertake such work. In particular, we focus on a substudy uniquely describing the response to exercise performed at scale with accompanying genetic information. [ABSTRACT FROM AUTHOR]

In the endothelium, ATP-sensitive potassium (KATP) channels are thought to couple cellular metabolism with membrane excitability, calcium entry, and endothelial mediator release. We hypothesized that endothelial KATP channels have a broad role protecting against high blood pressure and atherosclerosis. Endothelial-specific Kir6.1 KO mice (eKO) and eKO mice on an apolipoprotein E KO background were generated (A-eKO) to investigate the role of KATP channels in the endothelium. Basal blood pressure was not elevated in eKO mice. However, when challenged with a high-salt diet and the eNOS inhibitor L-NAME, eKO mice became more hypertensive than their littermate controls. In aorta, NO release at least partly contributes to the endothelium-dependent vasorelaxation induced by pinacidil. In A-eKO mice atherosclerotic plaque density was significantly greater than in their littermate controls when challenged with a high-fat diet, particularly in the aortic arch region. Levels of endothelial dysfunction markers were higher in eKO compared with WT mice; however, these were not significant for A-eKO mice compared with their littermate controls. Furthermore, decreased vascular reactivity was observed in the mesenteric arteries of A-eKO mice, but not in aorta when on a high-fat diet. Our data support a role for endothelial Kir6.1-containing KATP channels in the endothelial protection against environmental stressors: the maintenance of blood pressure homeostasis in response to high salt and endothelial integrity when challenged with a high-fat diet. [ABSTRACT FROM AUTHOR]

All Kv7 potassium channels require membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P) for their normal function and hence can be physiologically regulated by neurotransmitters and hormones that stimulate phosphoinositide hydrolysis. Recent mutational analysis indicates that a cluster of basic residues in the proximal C-terminus (K354/K358/R360/K362) is crucial for PI(4,5)P activation of cardiac Kv7.1 channels. Since this cluster is largely conserved in all Kv7 subunits, we tested whether homologous residues are also required for activation of Kv7.2 (a subunit of neuronal M-channels). We found that the mutation Kv7.2 (R325A) (corresponding to R360 in Kv7.1) reduced Kv7.2 current amplitude by ∼60 % ( P < 0.02) without change in voltage sensitivity and reduced the sensitivity of Kv7.2 channels to dioctanoyl-phosphatidylinositol-4,5-bisphosphate by ∼eightfold ( P < 0.001). Taking into account previous experiments (Zhang et al., Neuron 37:963-75, ) implicating Kv7.2 (H328), and since R325 and H328 are conserved in homologous positions in all other Kv7 channels, we suggest that this proximal C-terminal domain adjacent to the last transmembrane domain that contains R325 and H328 (in Kv7.2) might play a major role in the activation of all members of the Kv7 channel family by PI(4,5)P. [ABSTRACT FROM AUTHOR]

KCNE1 associates with the poreforming a-subunit KCNQI to generate the slow (/Ks) current in cardiac myocytes. Mutations in either KCNQ1 or KCNEI can alter the biophysical properties of `K. and mutations in KCNE1 underlie cases of long QT syndrome type 5 (LQT5). We previously investigated a mutation in KCNE 1, T58P1L59P, which causes severe attenuation of /Ks. However, how T58P/L59P acts to disrupt /Ks has not been determined. In this study, we investigate and compare the effects of T58PfL59P with three other LQT5 mutations (G52R, S74L, and R98W) on the biophysical properties of the current, trafficking of KCNQ1, and assembly of the /Ks channel. G52R and T58P/L59P produce currents that lack the kinetic behavior of /Ks. In contrast, S74L and R98W both produce 1K5-like currents but with rightward shifted voltage dependence of activation. All of the LQT5 mutants express protein robustly, and T58P1L59P and R98W cause modest, but significant, defects in the trafficking of KCNQ 1. Despite defects in trafficking, in the presence of KCNQ1, T58P/L59P and the other LQT5 mutants are present at the plasma membrane. Interestingly, in comparison to KCNEI and the other LQT5 mutants, T58P/L59P associates only weakly with KCNQ1. In conclusion, we identify the disease mechanisms for each mutation and reveal that T58P/L59P causes disease through a novel mechanism that involves defective /Ks complex assembly. [ABSTRACT FROM AUTHOR]

Aims: ATP sensitive K+ channels (KATP) sense adenine nucleotide concentrations and thus couple the metabolic state of the cell to membrane potential. The hetero-octameric complex of a sulphonylurea receptor (SUR2B) and an inwardly rectifying K+ channel (Kir6.1) and the corresponding native channel in smooth muscle are relatively insensitive to variations in intracellular ATP. Activation of these channels in blood vessels during hypoxia/ischaemia is thought to be mediated via hormonal regulation such as cellular adenosine release or the release of mediators from the endothelium. In contrast, intracellular ATP prominently inhibits Kir6.2 containing complexes, such as those present in cardiac myocytes. Thus, we investigated differences in the mechanism of metabolic regulation of Kir6.1 and Kir6.2 containing KATP channels. [ABSTRACT FROM PUBLISHER]

The open state of M(Kv7.2/7.3) potassium channels is maintained by membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). They can be closed on stimulating receptors that induce PI(4,5)P2 hydrolysis. In sympathetic neurons, closure induced by stimulating M1-muscarinic acetylcholine receptors (mAChRs) has been attributed to depletion of PI(4,5)P2, whereas closure by bradykinin B2-receptors (B2-BKRs) appears to result from formation of IP3 and release of Ca2+, implying that BKR stimulation does not deplete PI(4,5)P2. We have used a fluorescently tagged PI(4,5)P2-binding construct, the C-domain of the protein tubby, mutated to increase sensitivity to PI(4,5)P2 changes (tubby-R332H-cYFP), to provide an on-line read-out of PI(4,5)P2 changes in single living sympathetic neurons after receptor stimulation. We find that the mAChR agonist, oxotremorine-M (oxo-M), produces a near-complete translocation of tubby-R332H-cYFP into the cytoplasm, whereas bradykinin (BK) produced about one third as much translocation. However, translocation by BK was increased to equal that produced by oxo-M when synthesis of PI(4,5)P2 was inhibited by wortmannin. Further, wortmannin ‘rescued’ M-current inhibition by BK after Ca2+-dependent inhibition was reduced by thapsigargin. These results provide the first direct support for the view that BK accelerates PI(4,5)P2 synthesis in these neurons, and show that the mechanism of BKR-induced inhibition can be switched from Ca2+ dependent to PI(4,5)P2 dependent when PI(4,5)P2 synthesis is inhibited. [ABSTRACT FROM AUTHOR]

G protein-gated inwardly rectifying K[sup +] (Kir) channels are found in neurones, atrial myocytes, and endocrine cells and are involved in generating late inhibitory postsynaptic potentials, slowing the heart rate and inhibiting hormone release. They are activated by G proteincoupled receptors (GPCRs) via the inhibitory family of G protein, G[sub i/o], in a membrane-delimited fashion by the direct binding of Gβγ dimers to the channel complex. In this study we are concerned with the kinetics of deactivation of the cloned neuronal G protein-gated K[sup +] channel, Kir3.1 + 3.2A, after stimulation of a number of GPCRs. Termination of the channel activity on agonist removal is thought to solely depend on the intrinsic hydrolysis rate of the G protein α subunit. In this study we present data that illustrate a more complex behavior. We hypothesize that there are two processes that account for channel deactivation: agonist unbinding from the GPCR and GTP hydrolysis by the G protein α subunit. With some combinations of agonist/GPCR, the rate of agonist unbinding is slow and rate-limiting, and deactivation kinetics are not modulated by regulators of G protein-signaling proteins. In another group, channel deactivation is generally faster and limited by the hydrolysis rate of the G protein α subunit. G protein isoform and interaction with G protein-signaling proteins play a significant role with this group of GPCRs. [ABSTRACT FROM AUTHOR]

Central to the genesis of ventricular cardiac arrhythmia are variations in determinants of excitability. These involve individual ionic channels and transporters in cardiac myocytes but also tissue factors such as variable conduction of the excitation wave, fibrosis and source-sink mismatch. It is also known that in certain diseases and particularly the channelopathies critical events occur with specific stressors. For example, in hereditary long QT syndrome due to mutations in KCNQ1 arrhythmic episodes are provoked by exercise and in particular swimming. Thus not only is the static substrate important but also how this is modified by dynamic signalling events associated with common physiological responses. In this review, we examine the regulation of ventricular excitability by signalling pathways from a cellular and tissue perspective in an effort to identify key processes, effectors and potential therapeutic approaches. We specifically focus on the autonomic nervous system and related signalling pathways. [ABSTRACT FROM AUTHOR]

We have directly observed the trafficking and fusion of ion channel containing vesicles and monitored the release of individual ion channels at the plasma membrane of live mammalian cells using total internal reflection fluorescence microscopy. Proteins were fused in-frame with green or red fluorescent proteins and expressed at low level in HL-1 and HEK293 cells. Dual color imaging revealed that vesicle trafficking involved motorized movement along microtubules followed by stalling, fusion, and subsequent release of individual ion channels at the plasma membrane. We found that KCNQ1-KCNE1 complexes were released in batches of about 5 molecules per vesicle. To elucidate the properties of ion channel complexes at the cell membrane we tracked the movement of individual molecules and compared the diffusive behavior of two types of potassium channel complex (KCNQ1-KCNE1 and Kir6.2-SUR2A) to that of a G-protein coupled receptor, the Al adenosine receptor. Plots of mean squared displacement against time intervals showed that mobility depended on channel type, cell type, and temperature. Analysis of the mobility of wild type KCNQ1-KCNE1 complexes showed the existence of a significant immobile subpopulation and also a significant number of molecules that demonstrated periodic stalling of diffusive movements. This behavior was enhanced in cells treated with jasplakinolide and was abrogated in a C-terminal truncated form (KCNQ1(R518X)-KCNE1) of the protein. This mutant has been identified in patients with the long QT syndrome. We propose that KCNQ1-KCNE1 complexes interact intermittently with the actin cytoskeleton via the C-terminal region and this interaction may have a functional role. [ABSTRACT FROM AUTHOR]