The G-protein–gated K+ channel,IKACh, is required for regulation of pacemaker activity and recovery of resting heart rate after sympathetic stimulation

Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein–activated K⁺ current (I_KACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of I_KACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4^−/− mice), which codes for an integral subunit of the cardiac I_KACh channel. SAN pacemaker cells from Girk4^−/− mice completely lacked I_KACh. Loss of I_KACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4^−/− animals. Although the relative extent of heart rate regulation of Girk4^−/− mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological β-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4^−/− animals. We conclude that I_KACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct β-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for I_KACh in SAN physiology and heart rate regulation.     

Silencing GIRK4 expression in human atrial myocytes by adenovirus-delivered small hairpin RNA

GIRK4 has been shown to be a subunit of I_KACh, and the use of GIRK4 in human atrial myocytes to treat arrhythmia remains an important research pursuit. Adenovirus-delivered small hairpin RNA (shRNA) has been used to mediate gene knockdown in mouse cardiocytes, yet there is no information on the successful application of this technique in human cardiocytes. In the current study, we used a siRNA validation system to select the most efficient sequence for silencing GIRK4. To this end, adenovirus-delivered shRNA, which expresses this sequence, was used to silence GIRK4 expression in human atrial myocytes. Finally, the feasibility, challenges, and results of silencing GIRK4 expression were evaluated by RT-PCR, western blotting, and the voltage-clamp technique. The levels of mRNA and protein were depressed significantly in cells infected by adenovirus-delivered shRNA against GIRK4, approximately 86.3% and 51.1% lower than those cells infected by adenovirus-delivered nonsense shRNA, respectively. At the same time, I_KACh densities were decreased 53% by adenovirus-delivered shRNA against GIRK4. In summary, adenovirus-delivered shRNA against GIRK4 mediated efficient GIRK4 knockdown in human atrial myocytes and decreased I_KACh densities_. As such, these data indicated that adenovirus-delivered shRNA against GIRK4 is a potential tool for treating arrhythmia.     

Attenuation of Acetylcholine Activated Potassium Current (IKACh) by Simvastatin,Not Pravastatin in Mouse Atrial Cardiomyocyte: Possible Atrial Fibrillation Preventing Effects of Statin

Statins, 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, are associated with the prevention of atrial fibrillation (AF) by pleiotropic effects. Recent clinical trial studies have demonstrated conflicting results on anti-arrhythmia between lipophilic and hydrophilic statins. However, the underlying mechanisms responsible for anti-arrhythmogenic effects of statins are largely unexplored. In this study, we evaluated the different roles of lipophilic and hydrophilic statins (simvastatin and pravastatin, respectively) in acetylcholine (100 µM)-activated K⁺ current (I_KACh, recorded by nystatin-perforated whole cell patch clamp technique) which are important for AF initiation and maintenance in mouse atrial cardiomyocytes. Our results showed that simvastatin (1–10 µM) inhibited both peak and quasi-steady-state I_KACh in a dose-dependent manner. In contrast, pravastatin (10 µM) had no effect on I_KACh. Supplementation of substrates for the synthesis of cholesterol (mevalonate, geranylgeranyl pyrophosphate or farnesyl pyrophosphate) did not reverse the effect of simvastatin on I_KACh, suggesting a cholesterol-independent effect on I_KACh. Furthermore, supplementation of phosphatidylinositol 4,5-bisphosphate, extracellular perfusion of phospholipase C inhibitor or a protein kinase C (PKC) inhibitor had no effect on the inhibitory activity of simvastatin on I _KACh. Simvastatin also inhibits adenosine activated I_KACh, however, simvastatin does not inhibit I_KACh after activated by intracellular loading of GTP gamma S. Importantly, shortening of the action potential duration by acetylcholine was restored by simvastatin but not by pravastatin. Together, these findings demonstrate that lipophilic statins but not hydrophilic statins attenuate I_KACh in atrial cardiomyocytes via a mechanism that is independent of cholesterol synthesis or PKC pathway, but may be via the blockade of acetylcholine binding site. Our results may provide important background information for the use of statins in patients with AF.     

Short-Term Desensitization of Muscarinic K+ Current in the Heart

Acetylcholine (ACh) rapidly increases cardiac K⁺ currents (I_KACh) by activating muscarinic K⁺ (K_ACh) channels followed by a gradual amplitude decrease within seconds. This phenomenon is called short-term desensitization and its precise mechanism and physiological role are still unclear. We constructed a mathematical model for I_KACh to examine the conditions required to reconstitute short-term desensitization. Two conditions were crucial: two distinct muscarinic receptors (m₂Rs) with different affinities for ACh, which conferred an I_KACh response over a wide range of ACh concentrations, and two distinct K_ACh channels with different affinities for the G-protein βγ subunits, which contributed to reconstitution of the temporal behavior of I_KACh. Under these conditions, the model quantitatively reproduced several unique properties of short-term desensitization observed in myocytes: 1), the peak and quasi-steady states with 0.01–100 μM [ACh]; 2), effects of ACh preperfusion; and 3), recovery from short-term desensitization. In the presence of 10 μM ACh, the I_KACh model conferred recurring spontaneous firing after asystole of 8.9 s and 10.7 s for the Demir and Kurata sinoatrial node models, respectively. Therefore, two different populations of K_ACh channels and m₂Rs may participate in short-term desensitization of I_KACh in native myocytes, and may be responsible for vagal escape at nodal cells.     

Elevated Expression of the G-Protein-Activated Inwardly Rectifying Potassium Channel 2 (GIRK2) in Cerebellar Unipolar Brush Cells of a Down Syndrome Mouse Model

SUMMARY 1. Down syndrome (DS) arises from the presence of three copies of chromosome (Chr.) 21. Fine motor learning deficits found in DS from childhood to adulthood result from expression of extra genes on Chr. 21, however, it remains unclear which if any of these genes are the specific causes of the cognitive and motor dysfunction. DS cerebellum displays morphological abnormalities that likely contribute to the DS motor phenotype.2. The G-protein-activated inwardly rectifying potassium channel subunit 2 (GIRK2) is expressed in cerebellum and can shunt dendritic conductance and attenuate postsynaptic potentials. We have used an interbreeding approach to cross a genetic mouse model of DS (Ts65Dn) with Girk2 knockout mice and examined its relative expression level by quantitative real-time RT-PCR, Western blotting and immunohistochemistry.3. We report here for the first time that GIRK2 is expressed in unipolar brush cells, which are excitatory interneurons of the vestibulocerebellum and dorsal cochlear nucleus. Analysis of disomic-Ts65Dn/Girk2^(+/+/−) and heterozygous-Diploid/Girk2^(+/−) mice shows that GIRK2 expression in Ts65Dn lobule X follows gene dosage. The lobule X of Ts65Dn mice contain greater numbers of unipolar brush cells co-expressing GIRK2 and calretinin than the control mouse groups.4. These results demonstrate that gene triplication can impact specific cell types in the cerebellum. We hypothesize that GIRK2 overexpression will adversely affect cerebellar circuitry in Ts65Dn vestibulocerebellum and dorsal cochlear nucleus due to GIRK2 shunting properties and its effects on resting membrane potential.While Dr. Julius Axelrod’s impact on the development of Neuroscience was significant, one of his major contributions was made indirectly through the people close to him that he influenced. Being a Section Chief and colleague to Julie in the Laboratory of Clinical Science at the National Institute of Mental Health was one of the great honors of my life. It was always a joy observing humility, friendliness and concern of all problems big or small. At laboratory seminars it was a pleasure to watch Julie’s ideas and intuitions that often generated a tremendous amount of good science. He taught all of us how to be curious, incisive and imaginative, and above all to “keep it simple.” His delight in science was contagious. DMJ     

A Quantitative Model of the GIRK1/2 Channel Reveals That Its Basal and Evoked Activities Are Controlled by Unequal Stoichiometry of Gα and Gβγ

G protein-gated K⁺ channels (GIRK; Kir3), activated by Gβγ subunits derived from G_i/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (I_evoked) and neurotransmitter-independent basal (I_basal) GIRK activities are physiologically important, but mechanisms of I_basal and its relation to I_evoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that I_basal and I_evoked are interrelated: the extent of activation by neurotransmitter (activation index, R_a) is inversely related to I_basal. To unveil the underlying mechanisms, we have developed a quantitative model of GIRK1/2 function. We characterized single-channel and macroscopic GIRK1/2 currents, and surface densities of GIRK1/2 and Gβγ expressed in Xenopus oocytes. Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter. Our model accurately recapitulates I_basal and I_evoked in Xenopus oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gβγ and fully accounts for the inverse I_basal-R_a correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gβγ dimers are available for each GIRK1/2 channel. In contrast, available Gα_i/o decreases from ~2 to less than one Gα per channel as GIRK1/2''s density increases. The persistent Gβγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gβγ, consistent with recruitment to the cell surface of Gβγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gβγ but only 2 Gα_i/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gβγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gβγ and Gα.     

RGS6, but Not RGS4, Is the Dominant Regulator of G Protein Signaling (RGS) Modulator of the Parasympathetic Regulation of Mouse Heart Rate

Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M₂ muscarinic receptor (M₂R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K⁺ channel I_KACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4^−/−, Rgs6^−/−, and Rgs4^−/−:Rgs6^−/− mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M₂R-I_KACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M₂R-I_KACh signaling pathway in SAN cells to carbachol and a significant slowing of M₂R-I_KACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6^−/− mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6^−/− and Gβ5^−/− mice, suggest that the partial rescue of phenotypes in Rgs4^−/−:Rgs6^−/− mice is attributable to another R7 RGS protein whose influence on M₂R-I_KACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M₂R-I_KACh signaling in mice.     

Molecular cloning of the human gene STK10 encoding lymphocyte-oriented kinase, and comparative chromosomal mapping of the human, mouse, and rat homologues

 LOK is a new and unique member of the STE20 family with serine/threonine kinase activity, and its expression is restricted mostly to lymphoid cells in mice. We cloned the cDNA encoding the human homologue of LOK. The amino acid sequence deduced from the cDNA shows a high similarity to that of mouse LOK, with 88% identity as a whole. The kinase domains at the N-terminus and the coiled-coil regions at the C-terminus are particularly conserved, showing 98% and 93% identity, respectively. Western blot analysis with mouse LOK-specific antibody detected 130 000 M _r LOK proteins in human and rat lymphoid cell lines and tissues. The gene encoding the LOK (STK10/Stk10) gene was mapped by fluorescence in situ hybridization to chromosome 5q35.1 in human, chromosome 11A4 in mouse, and chromosome 10q12.3 in rat. By virtue of polymorphic CA repeats found in the 3' untranslated region of the mouse Stk10 gene, the Stk10 locus was further pinpointed to chromosome 11 between D11Mit53 and D11Mit84, using the intersubspecific backcross mapping panel. These results established STK10 as a new marker of human chromosome 5 to define the syntenic boundary of human chromosomes 5 and 16 on mouse chromosome 11. Received: 28 September 1998 / Revised: 2 November 1998     

Functional Expression and Characterization of G-protein-gated Inwardly Rectifying K+ Channels Containing GIRK3

The G-protein-gated inwardly rectifying K ⁺(GIRK) family of ion channels form functional Gβγ-sensitive channels as heteromultimers of GIRK1 and either the GIRK2 or GIRK4 subunits. However, the homologous mouse brain GIRK3 clone failed to express in the earliest reported functional experiments in Xenopus oocytes. We recloned the GIRK3 subunit from mouse brain and found that the new clone differed significantly from that originally reported. The functional aspects of GIRK3 were reinvestigated by expression in CHO cells. The single channel properties of GIRK1/GIRK3 were characterized and compared to those of the GIRK1/GIRK2 and GIRK1/GIRK4 channels. All three GIRK1/GIRKx combinations produced channels with nearly indistinguishable conductances and kinetics. The response of GIRK1/GIRK3 to Gβγ in the 1–47 nm range was examined and found to be indistinguishable from that of GIRK1/GIRK4 channels. We conclude that GIRK1, with either GIRK2, 3, or 4, gives rise to heteromultimeric channels with virtually identical conductances, kinetics, and Gβγ sensitivities. Received: 13 January 1999/Revised: 2 March 1999     

Inhibition of the cardiac inward rectifier potassium currents by KB-R7943

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea) was developed as a specific inhibitor of the sarcolemmal sodium–calcium exchanger (NCX) with potential experimental and therapeutic use. However, KB-R7943 is shown to be a potent blocker of several ion currents including inward and delayed rectifier K⁺ currents of cardiomyocytes. To further characterize KB-R7943 as a blocker of the cardiac inward rectifiers we compared KB-R7943 sensitivity of the background inward rectifier (I_K1) and the carbacholine-induced inward rectifier (I_KACh) currents in mammalian (Rattus norvegicus; rat) and fish (Carassius carassius; crucian carp) cardiac myocytes. The basal I_K1 of ventricular myocytes was blocked with apparent IC50-values of 4.6 × 10^− 6 M and 3.5 × 10^− 6 M for rat and fish, respectively. I_KACh was almost an order of magnitude more sensitive to KB-R7943 than I_K1 with IC₅₀-values of 6.2 × 10^− 7 M for rat and 2.5 × 10^− 7 M for fish. The fish cardiac NCX current was half-maximally blocked at the concentration of 1.9–3 × 10^− 6 M in both forward and reversed mode of operation. Thus, the sensitivity of three cardiac currents to KB-R7943 block increases in the order I_K1 ~ I_NCX < I_KACh. Therefore, the ability of KB-R7943 to block inward rectifier potassium currents, in particular I_KACh, should be taken into account when interpreting the data with this inhibitor from in vivo and in vitro experiments in both mammalian and fish models.     

The cardiac acetylcholine-activated,inwardly rectifying K+-channel subunit GIRK1 gives rise to an inward current induced by free oxygen radicals

Reactive oxygen species (ROS) play a crucial role in pathophysiology of the cardiovascular system. The present study was designed to analyze the redox sensitivity of G-protein-activated inward rectifier K⁺ (GIRK) channels, which control cardiac contractility and excitability. GIRK1 subunits were heterologously expressed in Xenopus laevis oocytes and the resulting K⁺ currents were measured with the two-electrode voltage clamp technique. Oxygen free radicals generated by the hypoxanthine/xanthine oxidase system led to a marked increase in the current through GIRK channels, termed superoxide-induced current (I_SO). Furthermore, I_SO did not depend on G-protein-dependent activation of GIRK currents by coexpressed muscarinic m₂-receptors, but could also be observed when no agonist was present in the bathing solution. Niflumic acid at a concentration of 0.5 mmol/l did not abolish I_SO, whereas 100 μmol/l Ba²⁺ attenuated I_SO completely. Catalase (10⁶ i.u./l) failed to suppress I_SO, whereas H₂O₂ concentration was kept close to zero, as measured by chemiluminescence. Hence, we conclude that O₂^•− or a closely related species is responsible for I_SO induction. Our results demonstrate a significant redox sensitivity of GIRK1 channels and suggest redox-activation of G-protein-activated inward rectifier K⁺ channels as a key mechanism in oxidative stress-associated cardiac dysfunction.     

Essential Role of the m2R-RGS6-IKACh Pathway in Controlling Intrinsic Heart Rate Variability

Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood.Here we examined the contribution of the m₂R-I_KACh intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m₂R-I_KACh signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the RGS6 gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m₂R-I_KACh signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis.     

Genome Organization of Human 48-kDa Oligosaccharyltransferase (DDOST)

The enzyme oligosaccharyltransferase (dolichyl-diphosphooligosaccharide–protein glycosyltransferase; EC 2.4.1.119) (DDOST) catalyzes the transfer of a high-mannose oligosaccharide (GlcNac₂Man₉Glc₃) from a dolichol-linked oligosaccharide donor (dolichol-P-GlcNac₂Man₉Glc₃) onto the asparagine acceptor site within an Asn-X-Ser/Thr consensus motif in nascent polypeptide chains across the membrane of the endoplasmic reticulum. We isolated mouse and human DDOST cDNAs from retinoic acid-treated mouse P19 EC cells and human NT-2 cells, respectively. DDOST mRNA is expressed intensely in heart and pancreas, but at lower levels in brain. Here we show that the human DDOST 48-kDa subunit gene (HGMW-approved symbol DDOST) is organized into 11 exons expanding about 9 kb. This DDOST subunit gene is localized on chromosome 1p36.1 by fluorescencein situhybridization analysis.     

Angiotensin II Type 1 Receptor-Mediated Electrical Remodeling in Mouse Cardiac Myocytes

We recently characterized an autocrine renin angiotensin system (RAS) in canine heart. Activation of Angiotensin II Type 1 Receptors (AT₁Rs) induced electrical remodeling, including inhibition of the transient outward potassium current I_to, prolongation of the action potential (AP), increased calcium entry and increased contractility. Electrical properties of the mouse heart are very different from those of dog heart, but if a similar system existed in mouse, it could be uniquely studied through genetic manipulations. To investigate the presence of a RAS in mouse, we measured APs and I_to in isolated myocytes. Application of angiotensin II (A2) for 2 or more hours reduced I_to magnitude, without affecting voltage dependence, and prolonged APs in a dose-dependent manner. Based on dose-inhibition curves, the fast and slow components of I_to (I_to,fast and I_K,slow) appeared to be coherently regulated by [A2], with 50% inhibition at an A2 concentration of about 400 nM. This very high K_0.5 is inconsistent with systemic A2 effects, but is consistent with an autocrine RAS in mouse heart. Pre-application of the microtubule destabilizing agent colchicine eliminated A2 effects on I_to and AP duration, suggesting these effects depend on intracellular trafficking. Application of the biased agonist SII ([Sar¹-Ile⁴-Ile⁸]A2), which stimulates receptor internalization without G protein activation, caused I_to reduction and AP prolongation similar to A2-induced changes. These data demonstrate AT₁R mediated regulation of I_to in mouse heart. Moreover, all measured properties parallel those measured in dog heart, suggesting an autocrine RAS may be a fundamental feedback system that is present across species.     

Investigation of pacemaker shift in the rabbit sinoatrial node using the optical mapping technique

Changes of the activation sequence in the rabbit sinoatrial node under the influence of low temperature and I _f selective blocker ivabradine have been studied using the optical mapping technique. Both factors caused a shift of the pacemaker within the sinoatrial node region. These results are compared with the data obtained recently in the investigation of pacemaker shift under the influence of cholinergic and adrenergic factors. Possible mechanisms of the pacemaker shift are discussed. The suppression of electric activity in the central part of the sinoatrial node during the action of acetylcholine, which is called cholinergic inexcitability, may be considered as one of the mechanisms of the pacemaker shift. It is shown that the main cause of cholinergic inexcitability is the activation of potassium acetylcholine-dependent current I _KACh.     

Partial Structure, Chromosome Localization, and Expression of the MouseIclnGene

The molecular identities of most volume-regulated ion channel proteins and putative regulatory elements are currently unknown. Recently, a role for a nucleotide-sensitive chloride conductance regulator, ICln, in the function of a ubiquitous volume-regulated chloride channel has been suggested. Here, we report the cloning of a fragment of the mouseIclngene and identification of probableIclnpseudogenes. The functionalIclngene was mapped independently to human chromosome 11q13.5–q14 and mouse chromosome 7 (50.3 cM). ICln mRNA was shown to be abundantly expressed and evenly distributed in all mouse tissues examined and at four stages of embryonic development, consistent with the proposed role of ICln in the regulation of a ubiquitous chloride channel.