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Abstract: The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, I KACh. However, it is not yet known if this response exhibits ‘fade’ or desensitization in the AVN, as established for the heart’s primary pacemaker – the sinoatrial node. In this study, acute activation of I KACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37°C. 0.1–1μM acetylcholine (ACh) rapidly activated a robust I KACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1μM). During sustained ACh exposure the elicited I KACh exhibited bi-exponential fade (τ f of 2.0s and τ s 76.9s at −120mV; 1μM ACh). 10nM ET-1 elicited a current similar to I KACh, which faded with a mono-exponential time-course (τ of 52.6s at −120mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization. [Copyright &y& Elsevier]

Abstract: G protein coupled inward rectifier K+ channels (GIRK) are activated by the Gβγ subunits of G proteins upon activation of G protein coupled receptors (GPCRs). Receptor-stimulated GIRK currents are known to possess a curious property, termed “agonist-dependent relaxation,” denoting a slow current increase upon stepping the membrane voltage from positive to negative potentials. Regulators of G protein signaling (RGS) proteins have earlier been implicated in this phenomenon since RGS coexpression was required for relaxation to be observed in heterologous expression systems. However, a recent study presented contrasting evidence that GIRK current relaxation reflects voltage sensitive agonist binding to the GPCR. The present study re-examined the role of RGS protein in agonist-dependent relaxation and found that RGS coexpression is not necessary for the relaxation phenomenon. However, RGS4 speeds up relaxation kinetics, allowing the phenomenon to be observed using shorter voltage steps. These findings resolve the controversy regarding the role of RGS protein vs. GPCR voltage sensitivity in mediating agonist-dependent relaxation of GIRK currents. [Copyright &y& Elsevier]

Abstract: The inward rectifier family of potassium (KCNJ) channels regulate vital cellular processes including cell volume, electrical excitability, and insulin secretion. Dysfunction of different isoforms have been linked to numerous diseases including Bartter’s, Andersen-Tawil, Smith-Magenis Syndromes, Type II diabetes mellitus, and epilepsy, making them important targets for therapeutic intervention. Using a family-based approach, we succeeded in expressing 10 of 11 human KCNJ channels tested in Saccharomyces cerevisiae. GFP-fusion proteins showed that these channels traffic correctly to the plasma-membrane suggesting that the protein is functional. A 2-step purification process can be used to purify the KCNJ channels to >95% purity in a mono-dispersed form. After incorporation into liposomes, 86Rb+ flux assays confirm the functionality of the purified proteins as inward rectifier potassium channels. [Copyright &y& Elsevier]

Abstract: The zebrafish has recently emerged as an excellent model for studies of heart development and regeneration. The physiology of the zebrafish heart has been suggested to resemble that of the human heart in many aspects, whereas, in contrast to mammals, the zebrafish has a remarkable ability to regenerate after heart injury. Thus, zebrafish have been proposed as a cost-effective model for genetic and pharmacological screens of factors affecting heart function and repair. However, realizing the full potential of the zebrafish heart as a model will require a better understanding of the electrophysiology of the adult zebrafish myocardium. Here, we characterize action potentials (APs) from intact adult atria and ventricles and find that the overall shape of zebrafish APs is similar to that of humans. We show that zebrafish, like most mammals, display functional acetylcholine-activated K+ channels in the atrium, but not in the ventricle. Furthermore, the zebrafish AP upstroke is dominated by Na+ channels, L-type Ca2+ channels contribute to the plateau phase and IKr channels are involved in repolarization. However, despite these similarities between zebrafish and mammalian electrophysiology, we also identified important differences. In particular, zebrafish display a robust T-type Ca2+ current in both atrial and ventricular cardiomyocytes. Interestingly, in most mammals T-type Ca2+ channels are only expressed in the developing heart or under pathophysiological conditions, indicating that adult zebrafish cardiomyocytes display a more immature phenotype. [Copyright &y& Elsevier]

Abstract: The inward rectifier current generated by Kir2.1 ion channel proteins is primarily responsible for the stable resting membrane potential in various excitable cell types, like neurons and myocytes. Tight regulation of Kir2.1 functioning prevents premature action potential formation and ensures optimal repolarization times. While Kir2.1 forward trafficking has been addressed in a number of studies, its degradation pathways are thus far unknown. Using three different lysosomal inhibitors, NH4Cl, chloroquine and leupeptin, we now demonstrate involvement of the lysosomal degradation pathway in Kir2.1 breakdown. Upon application of the inhibitors, increased steady state protein levels are detectable within few hours coinciding with intracellular granular Kir2.1 accumulation. Treatment for 24h with either chloroquine or leupeptin results in increased plasmamembrane originating inward rectifier current densities, while current–voltage characteristics remain unaltered. We conclude that the lysosomal degradation pathway contributes to Kir2.1 mediated inward rectifier current regulation. [Copyright &y& Elsevier]

Abstract: The functional significance of the electrophysiological properties of neural precursor cells (NPCs) was investigated using dissociated neurosphere-derived NPCs from the forebrain subventricular zone (SVZ) of adult mice. NPCs exhibited hyperpolarized resting membrane potentials, which were depolarized by the K+ channel inhibitor, Ba2+. Pharmacological analysis revealed two distinct K+ channel families: Ba2+-sensitive Kir channels and tetraethylammonium (TEA)-sensitive Kv (primarily KDR) channels. Ba2+ promoted mitogen-stimulated NPC proliferation, which was mimicked by high extracellular K+, whereas TEA inhibited proliferation. Based on gene and protein levels in vitro, we identified Kir4.1, Kir5.1 and Kv3.1 channels as the functional K+ channel candidates. Expression of these K+ channels was immunohistochemically found in NPCs of the adult mouse SVZ, but was negligible in neuroblasts. It therefore appears that expression of Kir and Kv (KDR) channels in NPCs and related changes in the resting membrane potential could contribute to NPC proliferation and neuronal lineage commitment in the neurogenic microenvironment. [Copyright &y& Elsevier]

Mutations of the KCNJ2 gene encoding the potassium channel Kir2.1 were previously shown to cause Andersen''s syndrome (AS), a multisystem disease manifesting with developmental abnormalities, cardiac arrhythmias and periodic paralyses. We conducted a search for KCNJ2 mutations among 188 unrelated patients suspected to have long QT syndrome (LQTS). The screening was performed by denaturing high-performance liquid chromatography (dHPLC) and DNA sequencing. Two novel mutations of the KCNJ2 gene were detected: a missense threonine to alanine mutation (T75A) in the N-terminal region (family 1) and an in-frame deletion of two amino acids (ΔFQ163-164) in the M2 transmembrane region (family 2). In addition, a previously described silent polymorphism C1146T was detected. In family 1, some of the affected family members had a history of periodic muscle weakness characteristic of AS, but no dysmorphic features. The mean QTc interval of the affected members were 444 ± 24 ms (family 1, n=7) and 456 ± 8 ms (family 2, n=2). The mutations affect functionally important regions of the KCNJ2 channel protein: upon injection of the Xenopus oocytes with the wild type and mutant KCNJ2 constructs, the channel proteins were correctly synthesized and localized to the cell surface, but no measurable inward K+ current could be detected for the mutant KCNJ2 constructs. In conclusion, we report two novel loss-of-function mutations of the KCNJ2 channel, affecting different domains of the channel protein. Mutations of the KCNJ2 gene should be considered in genetic subclassification of LQTS patients, even in the absence of overt manifestations of AS. [Copyright &y& Elsevier]

We tested whether the absence of an inward rectifier channel in slow skeletal muscle fibers of the frog is regulated by innervation. Normal and denervated slow fibers were identified according to their passive electrical properties. In current-clamp experiments, anomalous rectification was quantified as the ratio of effective resistances for hyperpolarizing and depolarizing pulses. In isotonic potassium solution, this ratio was 0.45 ± 0.1 (n = 14) for twitch fibers, whereas slow fibers displayed linear behavior [ratio = 1.0 ± 0.05 (n = 15)]. However, denervated slow fibers showed anomalous rectification (ratio, 0.48 ± 0.07; n = 5). This finding was supported by voltage-clamp experiments in which denervated slow fibers displayed (1) an inward rectifier current during hyperpolarizing pulses, (2) an increase in this current when [K+]o was increased, and (3) a current inhibition after application of Ba2+. These results suggest that frog slow fibers, which normally do not possess inward rectifier channels, can express them after denervation. [Copyright &y& Elsevier]

The subcellular distribution of ATP-sensitive potassium (KATP) channel subunits in rat-isolated ventricular myocytes was investigated using a panel of subunit-specific antisera. Kir6.1 subunits were associated predominantly with myofibril structures and were co-localized with the mitochondrial marker MitoFluor red (correlation coefficient (cc) = 0.63 ± 0.05). Anti-Kir6.1 antibodies specifically recognized a polypeptide of 48 kDa in mitochondrial membrane fractions consistent with the presence of Kir6.1 subunits in this organelle. Both Kir6.2 and SUR2A subunits were distributed universally over the sarcolemma. Lower-intensity antibody-associated immunofluorescence was detected intracellularly, which was correlated with the distribution of MitoFluor red in both cases (cc, Kir6.2, 0.56 ± 0.05; SUR2A, 0.61 ± 0.06). A polypeptide of 40 kDa was recognized by anti-Kir6.2-subunit antibodies in western blots of both microsomal and mitochondrial membrane fractions consistent with the presence of this subunit in the sarcolemma and mitochondria. Similarly, SUR2A and SUR2B subunits were detected in western blots of microsomal membrane proteins consistent with a sarcolemmal localization for these polypeptides. SUR2B subunits were shown in confocal microscopy to co-localize strongly with t-tubules (cc, 0.81 ± 0.05). Together, the results indicate that Kir6.2 and SUR2A subunits predominate in the sarcolemma of ventricular myocytes consistent with a Kir6.2/SUR2A-subunit combination in the sarcolemmal KATPchannel. Kir6.1, Kir6.2 and SUR2A subunits were demonstrated in mitochondria. Combinations of these subunits would not explain the reported pharmacology of the mitochondrial KATP channel (Mol Pharmacol 59 (2001) 225) suggesting the possibility of further unidentified components of this channel. [Copyright &y& Elsevier]