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ABSTRACT: Cardiac ventricular myocytes possess an extensive t-tubular system that facilitates the propagation of membrane potential across the cell body. It is well established that ionic currents at the restricted t-tubular space may lead to significant changes in ion concentrations, which, in turn, may affect t-tubular membrane potential. In this study, we used the whole cell patch-clamp technique to study accumulation and depletion of t-tubular potassium by measuring inward rectifier potassium tail currents (I(K1,tail)), and inward rectifier potassium current (I(K1)) "inactivation". At room temperatures and in the absence of Mg(2+) ions in pipette solution, the amplitude of I(K1,tail) measured ~10 min after the establishment of whole cell configuration was reduced by ~18%, but declined nearly twofold in the presence of 1 mM cyanide. At ~35°C I(K1,tail) was essentially preserved in intact cells, but its amplitude declined by ~85% within 5 min of cell dialysis, even in the absence of cyanide. Intracellular Mg(2+) ions played protective role at all temperatures. Decline of I(K1,tail) was accompanied by characteristic changes in its kinetics, as well as by changes in the kinetics of I(K1) inactivation, a marker of depletion of t-tubular K(+). The data point to remodeling of t tubules as the primary reason for the observed effects. Consistent with this, detubulation of myocytes using formamide-induced osmotic stress significantly reduced I(K1,tail), as well as the inactivation of inward I(K1). Overall, the data provide strong evidence that changes in t tubule volume/structure may occur on a short time scale in response to various types of stress.
AJP Heart and Circulatory Physiology 09/2011; 301(5):H1984-95. · 3.71 Impact Factor
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ABSTRACT: Kir2 subunits form channels that underlie classical strongly inwardly rectifying potassium currents. While homomeric Kir2 channels display a number of distinct and physiologically important properties, the functional properties of heteromeric Kir2 assemblies, as well as the stoichiometries and the arrangements of Kir2 subunits in native channels, remain largely unknown. Therefore, we have implemented a concatemeric approach, whereby all four cloned Kir2 subunits were linked in tandem, in order to study the effects of Kir2.1 and Kir2.2 heteromerization on properties of the resulting channels. Kir2.2 subunits contributed stronger to single-channel conductance than Kir2.1 subunits, and channels containing two or more Kir2.2 subunits displayed conductances indistinguishable from that of a Kir2.2 homomeric channel. In contrast, single-channel kinetics was a more discriminating property. The open times were significantly shorter in Kir2.2 channels compared with Kir2.1 channels and decreased nearly proportionally to the number of Kir2.2 subunits in the heteromeric channel. Similarly, the sensitivity to block by barium also depended on the proportions of Kir2.1 to Kir2.2 subunits. Overall, the results showed that Kir2.1 and Kir2.2 subunits exert neither a dominant nor an anomalous effect on any of the properties of heteromeric channels. The data highlight opportunities and challenges of using differential properties of Kir2 channels in deciphering the subunit composition of native inwardly rectifying potassium currents.
Pflügers Archiv - European Journal of Physiology 10/2010; 460(5):839-49. · 4.46 Impact Factor
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ABSTRACT: Cardiac I(K1) and I(KACh) are the major potassium currents displaying classical strong inward rectification, a unique property that is critical for their roles in cardiac excitability. In the last 15 years, research on I(K1) and I(KACh) has been propelled by the cloning of the underlying inwardly rectifying potassium (Kir) channels, the discovery of the molecular mechanism of strong rectification and the linking of a number of disorders of cardiac excitability to defects in genes encoding Kir channels. Disease-causing mutations in Kir genes have been shown experimentally to affect one or more of the following channel properties: structure, assembly, trafficking, and regulation, with the ultimate effect of a gain- or a loss-of-function of the channel. It is now established that I(K1) and I(KACh) channels are heterotetramers of Kir2 and Kir3 subunits, respectively. Each homomeric Kir channel has distinct biophysical and regulatory properties, and individual Kir subunits often display different patterns of regional, cellular, and membrane distribution. These differences are thought to underlie important variations in the physiological properties of I(K1) and I(KACh). It has become increasingly clear that the contribution of I(K1) and I(KACh) channels to cardiac electrical activity goes beyond their long recognized role in the stabilization of resting membrane potential and shaping the late phase of action potential repolarization in individual myocytes but extends to being critical elements determining the overall electrical stability of the heart.
Journal of Molecular and Cellular Cardiology 09/2009; 48(1):45-54. · 5.17 Impact Factor
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Jiumei Chen,
Jessica A Hessler,
Krishna Putchakayala,
Brian K Panama,
Damian P Khan,
Seungpyo Hong,
Douglas G Mullen,
Stassi C Dimaggio,
Abhigyan Som,
Gregory N Tew, Anatoli N Lopatin,
James R Baker,
Mark M Banaszak Holl,
Bradford G Orr
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ABSTRACT: It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes.
The Journal of Physical Chemistry B 08/2009; 113(32):11179-85. · 3.70 Impact Factor
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Sharlene M Day,
Pierre Coutu,
Wang Wang,
Todd Herron,
Immanuel Turner,
Michael Shillingford,
Nathan C Lacross,
Kimber L Converso,
Lin Piao,
Jingdong Li, Anatoli N Lopatin,
Joseph M Metzger
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ABSTRACT: Relaxation abnormalities are prevalent in heart failure and contribute to clinical outcomes. Disruption of Ca2+ homeostasis in heart failure delays relaxation by prolonging the intracellular Ca2+ transient. We sought to speed cardiac relaxation in vivo by cardiac-directed transgene expression of parvalbumin (Parv), a cytosolic Ca2+ buffer normally expressed in fast-twitch skeletal muscle. A key feature of Parv's function resides in its Ca2+/Mg2+ binding affinities that account for delayed Ca2+ buffering in response to the intracellular Ca2+ transient. Cardiac Parv expression decreased sarcoplasmic reticulum Ca2+ content without otherwise altering intracellular Ca2+ homeostasis. At high physiological mouse heart rates in vivo, Parv modestly accelerated relaxation without affecting cardiac morphology or systolic function. Ex vivo pacing of the isolated heart revealed a marked heart rate dependence of Parv's delayed Ca2+ buffering effects on myocardial performance. As the pacing frequency was lowered (7 to 2.5 Hz), the relaxation rates increased in Parv hearts. However, as pacing rates approached the dynamic range in humans, Parv hearts demonstrated decreased contractility, consistent with Parv buffering systolic Ca2+. Mathematical modeling and in vitro studies provide the underlying mechanism responsible for the frequency-dependent fractional Ca2+ buffering action of Parv. Future studies directed toward refining the dose and frequency-response relationships of Parv in the heart or engineering novel Parv-based Ca2+ buffers with modified Mg2+ and Ca2+ affinities to limit systolic Ca2+ buffering may hold promise for the development of new therapies to remediate relaxation abnormalities in heart failure.
Physiological Genomics 06/2008; 33(3):312-22. · 2.73 Impact Factor
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ABSTRACT: Previous studies have shown that cardiac inward rectifier potassium current (I(K1)) channels are heteromers of distinct Kir2 subunits and suggested that species- and tissue-dependent expression of these subunits may underlie variability of I(K1). In this study, we investigated the contribution of the slowly activating Kir2.3 subunit and free intracellular polyamines (PAs) to variability of I(K1) in the mouse heart. The kinetics of activation was measured in Kir2 concatemeric tetramers with known subunit stoichiometry. Inclusion of only one Kir2.3 subunit to a Kir2.1 channel led to an approximate threefold slowing of activation kinetics, with greater slowing on subsequent additions of Kir2.3 subunits. Activation kinetics of I(K1) in both ventricles and both atria was found to correspond to fast-activating Kir2.1/Kir2.2 channels, suggesting no major contribution of Kir2.3 subunits. In contrast, I(K1) displayed significant variation in both the current density and inward rectification, suggesting involvement of intracellular PAs. The total levels of PAs were similar across the mouse heart. Measurements of the free intracellular PAs in isolated myocytes, using transgenically expressed Kir2.1 channels as PA sensors, revealed "microheterogeneity" of I(K1) rectification as well as lower levels of free PAs in atrial myocytes compared with ventricular cells. These findings provide a quantitative explanation for the regional heterogeneity of I(K1).
AJP Heart and Circulatory Physiology 01/2008; 293(6):H3558-67. · 3.71 Impact Factor
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Luis F Lopez-Santiago,
Laurence S Meadows,
Sara J Ernst,
Chunling Chen,
Jyoti Dhar Malhotra,
Dyke P McEwen,
Audrey Speelman,
Jeffrey L Noebels,
Sebastian K G Maier, Anatoli N Lopatin,
Lori L Isom
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ABSTRACT: In neurons, voltage-gated sodium channel beta subunits regulate the expression levels, subcellular localization, and electrophysiological properties of sodium channel alpha subunits. However, the contribution of beta subunits to sodium channel function in heart is poorly understood. We examined the role of beta1 in cardiac excitability using Scn1b null mice. Compared to wildtype mice, electrocardiograms recorded from Scn1b null mice displayed longer RR intervals and extended QT(c) intervals, both before and after autonomic block. In acutely dissociated ventricular myocytes, loss of beta1 expression resulted in a approximately 1.6-fold increase in both peak and persistent sodium current while channel gating and kinetics were unaffected. Na(v)1.5 expression increased in null myocytes approximately 1.3-fold. Action potential recordings in acutely dissociated ventricular myocytes showed slowed repolarization, supporting the extended QT(c) interval. Immunostaining of individual myocytes or ventricular sections revealed no discernable alterations in the localization of sodium channel alpha or beta subunits, ankyrin(B), ankyrin(G), N-cadherin, or connexin-43. Together, these results suggest that beta1 is critical for normal cardiac excitability and loss of beta1 may be associated with a long QT phenotype.
Journal of Molecular and Cellular Cardiology 12/2007; 43(5):636-47. · 5.17 Impact Factor
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ABSTRACT: The role of the cardiac current Ik1 in arrhythmogenesis remains highly controversal. To gain further insights into the mechanisms of IK1 involvement in cardiac excitability, we studied the susceptibility of transgenic mice with altered IK1 to arrhythmia during various pharmacological and physiological challenges. Arrhythmogenesis was studied in transgenic mice expressing either dominant negative Kir2.1-AAA or wild type Kir2.1 subunits in the heart, models of IK1 suppression (AAA-TG) and up-regulation (WT-TG), respectively. Under normal conditions, both anesthetized wild type (WT) and AAA-TG mice did not display any spontaneous arrhythmias. In contrast,WT-TG mice displayed numerous arrhythmias of various types. In isolated hearts, the threshold concentration for halothane-induced ventricular tachycardias (VT) was increased to 167% [corrected] in the AAA-TG and decreased to 54% [corrected] in WT-TG hearts when compared to WT hearts. The number of PVCs induced by AV node ablation combined with hypokalemia was reduced in AAA-TG hearts and increased in WT-TG mice. After AV node ablation AAA-TG hearts were more tolerant, and WT-TG less tolerant to isoproterenol- induced arrhythmias than WT hearts. Analysis of monophasic action potentials in isolated hearts shows a significant reduction in the dispersion of action potential repolarization in mice with suppressed IK1. The data strongly support the hypothesis that in the mouse heart upregulation of IK1 is proarrhythmic, and that under certain conditions IK1 blockade in cardiac myocytes may be a potentially useful antiarrhythmic strategy.
Archiv für Kreislaufforschung 10/2007; 102(5):416-28. · 7.35 Impact Factor
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ABSTRACT: It is established that prolonged hypoxia leads to activation of K(ATP) channels and action potential (AP) shortening, but the mechanisms behind the early phase of metabolic stress remain controversial. Under normal conditions IK1 channels are constitutively active while K(ATP) channels are closed. Therefore, early changes in IK1 may underlie early AP shortening. This hypothesis was tested using transgenic mice with suppressed IK1 (AAA-TG). In isolated AAA-TG hearts AP shortening was delayed by approximately 24 s compared to WT hearts. In WT ventricular myocytes, blocking oxidative phosphorylation with 1 mM cyanide (CN; 28 degrees C) led to a 29% decrease in APD90 within approximately 3-5 min. The effect of CN was reversed by application of 100 microM Ba2+, a selective blocker of IK1, but not by 10 microM glybenclamide, a selective blocker of KATP channels. Accordingly, voltage-clamp experiments revealed that both CN and true hypoxia lead to early activation of IK1. In AAA-TG myocytes, neither CN nor glybenclamide or Ba2+ had any effect on AP. Further experiments showed that buffering of intracellular Ca2+ with 20 mM BAPTA prevented IK1 activation by CN, although CN still caused a 54% increase in IK1 in a Ca2+ -free bath solution. Importantly, both (i) 20 microM ruthenium red, a selective inhibitor of SR Ca2+ -release, and (ii) depleting SR by application of 10 microM ryanodine+1 mM caffeine, abolished the activation of IK1 by CN. The above data strongly argue that in the mouse heart IK1, not KATP, channels are responsible for the early AP shortening during hypoxia.
Journal of Molecular and Cellular Cardiology 08/2007; 43(1):27-38. · 5.17 Impact Factor
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ABSTRACT: Sarcolemmal ATP-sensitive potassium (K(ATP)) channels are activated after pathological depletion of intracellular ATP, unlike their pancreatic beta-cell counterparts, which dynamically regulate membrane excitability in response to changes in blood glucose. We recently engineered a series of transgenic (TG) mice overexpressing an ATP-insensitive inward rectifying K(+) channel protein (Kir)6.2 mutant (Kir6.2[DeltaN30,K185Q]) or the accessory sulfonylurea receptor (SUR)2A (FLAG-SUR2A) or SUR1 (FLAG-SUR1) subunits of the K(ATP) channel, under transcriptional control of the alpha-myosin heavy chain promoter. In the present study, we generated double transgenic (DTG) animals overexpressing both Kir6.2[DeltaN30,K185Q] and FLAG-SUR1 or FLAG-SUR2A and examined the effects on cardiac excitability in vivo. No animals expressing both FLAG-SUR1 and Kir6.2[DeltaN30,K185Q] transgenes at a high level were obtained. DTG mice expressing one transgene at a high level and the other at a lower level are born, but they die prematurely. Electrocardiographic analysis of both anesthetized and conscious animals revealed a constellation of arrhythmias in DTG animals, but not in wild-type or single TG littermates. The proarrhythmic effect of the transgene combination is intrinsic to the myocardium, since it persists in isolated hearts. Importantly, this effect is specific for SUR1-expressing DTG animals: DTG animals expressing both Kir6.2[DeltaN30,K185Q] and FLAG-SUR2A at high levels exhibit neither impaired survival nor increased arrhythmia frequency, even with both subunits expressed at high levels. In demonstrating the profound arrhythmic consequences of K(ATP) channels comprised of SUR1 and Kir6.2[DeltaN30,K185Q] in the myocardium specifically, the results highlight the critical differential activation of SUR1 versus SUR2A, and indicate that expression of hyperactive K(ATP) in the heart is likely to be proarrhythmic.
AJP Heart and Circulatory Physiology 08/2007; 293(1):H836-45. · 3.71 Impact Factor
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ABSTRACT: G protein-coupled receptors play a pivotal role in regulating cardiac automaticity. Their function is controlled by regulator of G protein signaling (RGS) proteins acting as GTPase-activating proteins for Galpha subunits to suppress Galpha(i) and Galpha(q) signaling. Using knock-in mice in which Galpha(i2)-RGS binding and negative regulation are disrupted by a genomic Galpha(i2)G184S (GS) point mutation, we recently (Fu Y, Huang X, Zhong H, Mortensen RM, D'Alecy LG, Neubig RR. Circ Res 98: 659-666, 2006) showed that endogenous RGS proteins suppress muscarinic receptor-mediated bradycardia. To determine whether this was due to direct regulation of cardiac pacemakers or to alterations in the central nervous system or vascular responses, we examined isolated, perfused hearts. Isoproterenol-stimulated beating rates of heterozygote (+/GS) and homozygote (GS/GS) hearts were significantly more sensitive to inhibition by carbachol than were those of wild type (+/+). Even greater effects were seen in the absence of isoproterenol; the potency of muscarinic-mediated bradycardia was enhanced fivefold in GS/GS and twofold in +/GS hearts compared with +/+. A(1)-adenosine receptor-mediated bradycardia was unaffected. In addition to effects on the sinoatrial node, +/GS and GS/GS hearts show significantly increased carbachol-induced third-degree atrioventricular (AV) block. Atrial pacing studies demonstrated an increased PR interval and AV effective refractory period in GS/GS hearts compared with +/+. Thus loss of the inhibitory action of endogenous RGS proteins on Galpha(i2) potentiates muscarinic inhibition of cardiac automaticity and conduction. The severe carbachol-induced sinus bradycardia in Galpha(i2)G184S mice suggests a possible role for alterations of Galpha(i2) or RGS proteins in sick sinus syndrome and pathological AV block.
AJP Heart and Circulatory Physiology 06/2007; 292(5):H2532-9. · 3.71 Impact Factor
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ABSTRACT: Previous studies have suggested an important role for the inward rectifier K+ current (I K1) in stabilizing rotors responsible for ventricular tachycardia (VT) and fibrillation (VF). To test this hypothesis, we used a line of transgenic mice (TG) overexpressing Kir 2.1-green fluorescent protein (GFP) fusion protein in a cardiac-specific manner. Optical mapping of the epicardial surface in ventricles showed that the Langendorff-perfused TG hearts were able to sustain stable VT/VF for 350 +/- 1181 s at a very high dominant frequency (DF) of 44.6 +/- 4.3 Hz. In contrast, tachyarrhythmias in wild-type hearts (WT) were short-lived (3 +/- 9 s), and the DF was 26.3 +/- 5.2 Hz. The stable, high frequency, reentrant activity in TG hearts slowed down, and eventually terminated in the presence of 10 mum Ba2+, suggesting an important role for I K1. Moreover, by increasing I K1 density in a two-dimensional computer model having realistic mouse ionic and action potential properties, a highly stable, fast rotor (approximately 45 Hz) could be induced. Simulations suggested that the TG hearts allowed such a fast and stable rotor because of both greater outward conductance at the core and shortened action potential duration in the core vicinity, as well as increased excitability, in part due to faster recovery of Na+ current. The latter resulted in a larger rate of increase in the local conduction velocity as a function of the distance from the core in TG compared to WT hearts, in both simulations and experiments. Finally, simulations showed that rotor frequencies were more sensitive to changes (doubling) in I K1, compared to other K+ currents. In combination, these results provide the first direct evidence that I K1 up-regulation in the mouse heart is a substrate for stable and very fast rotors.
The Journal of Physiology 02/2007; 578(Pt 1):315-26. · 4.72 Impact Factor
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ABSTRACT: Recent studies have shown that Kir2 channels display differential sensitivity to intracellular polyamines, and have raised a number of questions about several properties of inward rectification important to the understanding of their physiological roles. In this study, we have carried out a detailed characterization of steady-state and kinetic properties of block of Kir2.1-3 channels by spermine. High-resolution recordings from outside-out patches showed that in all Kir2 channels current-voltage relationships display a 'crossover' effect upon change in extracellular K+. Experiments at different concentrations of spermine allowed for the characterization of two distinct shallow components of rectification, with the voltages for half-block negative (V1(1/2)) and positive (V2(1/2)) to the voltage of half-block for the major steep component of rectification (V0(1/2)). While V1(1/2) and V2(1/2) voltages differ significantly between Kir2 channels, they were coupled to each other according to the equation V1(1/2)-V2(1/2) = constant, strongly suggesting that similar structures may underlie both components. In Kir2.3 channels, the V2(1/2) was approximately 50 mV positive to V0(1/2), leading to a pattern of outward currents distinct from that of Kir2.1 and Kir2.2 channels. The effective valency of spermine block (Z0) was highest in Kir2.2 channels while the valencies in Kir2.1 and Kir2.3 channels were not significantly different. The voltage dependence of spermine unblock was similar in all Kir2 channels, but the rates of unblock were approximately 7-fold and approximately 16-fold slower in Kir2.3 channels than those in Kir2.1 and Kir2.2 when measured at high and physiological extracellular K+, respectively. In all Kir2 channels, the instantaneous phase of activation was present. The instantaneous phase was difficult to resolve at high extracellular K+ but it became evident and accounted for nearly 30-50% of the total current when recorded at physiological extracellular K+. In conclusion, the data are consistent with the universal mechanism of rectification in Kir2 channels, but also point to significant, and physiologically important, quantitative differences between Kir2 isoforms.
The Journal of Physiology 04/2006; 571(Pt 2):287-302. · 4.72 Impact Factor
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ABSTRACT: The lack of pathological consequences of cardiac ATP-sensitive potassium channel (K(ATP)) channel gene manipulation is in stark contrast to the effect of similar perturbations in the pancreatic beta-cell. Because the pancreatic and cardiac channel share the same pore-forming subunit (Kir6.2), the different effects of genetic manipulation likely reflect, at least in part, the tissue-specific expression of the regulatory subunit (SUR1 in pancreas vs. SUR2A in heart) of the bipartite channel complex. To examine this, we have generated transgenic (TG) mice that overexpress epitope-tagged SUR1 or SUR2A under the transcriptional control of the alpha-myosin heavy chain promoter. Western blot and real time RT-PCR analysis confirm transgene expression in the heart, and variable levels of SUR1 RNA and protein, in 16 viable founder lines. Surprisingly, activation of channels by either pharmacological agents (diazoxide and pinacidil) or metabolic inhibitors (oligomycin and 2-deoxyglucose) reveals a suppression of total K(ATP) conductance in high expressing TG mice. Moreover, K(ATP) channel activity was significantly reduced in excised cardiac patches from TG myocytes that overexpress either SUR1 or SUR2A. Using a recombinant cell system, we show that overexpression of either SUR1 or Kir6.2 suppresses the functional expression of K(ATP) from optimized dimeric SUR1-Kir6.2. Thus, the graded effect of SUR1 expression in the intact heart appears to demonstrate an in vivo requirement for 1:1 expression ratio of Kir6.2 and SURx.
Journal of Molecular and Cellular Cardiology 11/2005; 39(4):647-56. · 5.17 Impact Factor
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ABSTRACT: To assess the functional significance of upregulation of the cardiac current (IK1), we have produced and characterized the first transgenic (TG) mouse model of IK1 upregulation. To increase IK1 density, a pore-forming subunit of the Kir2.1 (green fluorescent protein-tagged) channel was expressed in the heart under control of the alpha-myosin heavy chain promoter. Two lines of TG animals were established with a high level of TG expression in all major parts of the heart: line 1 mice were characterized by 14% heart hypertrophy and a normal life span; line 2 mice displayed an increased mortality rate, and in mice < or =1 mo old, heart weight-to-body weight ratio was increased by >100%. In adult ventricular myocytes expressing the Kir2.1-GFP subunit, IK1 conductance at the reversal potential was increased approximately 9- and approximately 10-fold in lines 1 and 2, respectively. Expression of the Kir2.1 transgene in line 2 ventricular myocytes was heterogeneous when assayed by single-cell analysis of GFP fluorescence. Surface ECG recordings in line 2 mice revealed numerous abnormalities of excitability, including slowed heart rate, premature ventricular contractions, atrioventricular block, and atrial fibrillation. Line 1 mice displayed a less severe phenotype. In both TG lines, action potential duration at 90% repolarization and monophasic action potential at 75-90% repolarization were significantly reduced, leading to neuronlike action potentials, and the slow phase of the T wave was abolished, leading to a short Q-T interval. This study provides a new TG model of IK1 upregulation, confirms the significant role of IK1 in cardiac excitability, and is consistent with adverse effects of IK1 upregulation on cardiac electrical activity.
AJP Heart and Circulatory Physiology 01/2005; 287(6):H2790-802. · 3.71 Impact Factor
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ABSTRACT: The inward rectifier potassium current in the heart, I(K1), has been suggested to play a significant role in cardiac excitability by contributing to the late phase of action potential (AP) repolarization and the stabilization of resting potential. To further assess the role of I(K1) in cardiac excitability we have produced transgenic mice expressing a dominant-negative subunit of the Kir2.1 channel, a major molecular determinant of I(K1) in the heart, and studied the effects of I(K1) suppression on major potassium currents, APs and the overall electrical activity of the heart. Kir2.1 channel subunits with a mutated signature sequence (AAA for GYG substitution) were expressed in the heart under control of the alpha-myosin heavy chain promoter. Two lines of transgenic mice were established, both expressing high levels of Kir2.1-AAA-GFP (GFP, green fluorescent protein) subunits in all major parts of the heart. In ventricular myocytes isolated from transgenic mice, I(K1) was reduced by 95% in both lines, leading to a significant prolongation of APs. Surface ECG recordings from anesthetized transgenic mice revealed significant changes in key parameters of excitability, including prolongation of QRS complexes and QT intervals. This study confirms the significant role of I(K1) in control of AP repolarization and major ECG intervals in the intact heart.
Journal of Molecular and Cellular Cardiology 05/2003; 35(4):367-78. · 5.17 Impact Factor
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ABSTRACT: A detailed mechanistic understanding of ion-channel function has been achieved over the last 40 years, propelled in the last
15 years by advances in patch-clamp technology and most recently by determination of channel structures and their manipulation.
From the pharmacological perspective, perhaps as important as knowing how channels work, is knowing where they are—both where
in tissues, and ultimately, where in individual cells. Because ion channels are integral membrane proteins, their localization
can be studied using established general approaches. Immunofluorescent labeling has been widely used to map cloned Ca2+ (1), Na+(2), and K+ (3) channels, for which antibodies have been raised against the channel protein. Different variations of channel immunolabeling,
including immunogold (4) (Na+ channels) or radiolabel staining are available in this arsenal. A less general but specific technique commonly used with
ion channels is to use high-affinity toxins, either fluorescently or radioactively labeled, that specifically bind to the
channel pore or regulatory subunits. Among these are scorpion toxins to map Na+ channels (5), Σ-conotoxin to map Ca2+ channels (6), α-dendrotoxin to map voltage-gated channels (7) and α-bungarotoxin (8) to map nicotinic ACh receptors. Such techniques, in addition to being expensive and time consuming, generally require tissue
fixation, and are not amenable to living cells. Green fluorescent protein (GFP) (9) based technology is now revolutionizing the localization of proteins, including ion channels (see Part 2 of this book). Attaching GFP to N or C termini of the protein of interest permits visualization in living or fixed
preparations, generally without significantly affecting channel function.
12/2000: pages 311-325;
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ABSTRACT: The mechanism of spermidine release from Xenopus oocytes was examined by measuring release of radioactive [3H]spermidine under different ionic conditions, and under voltage-clamp. In normal solution (2 mM K), the efflux rate is less than 1% per hour, and is stimulated 2-fold by inclusion of Ca (1 mM) in the incubation medium. Spermidine efflux is stimulated 10-fold in high [K] (KD98) solution. In KD98 solution, efflux is strongly inhibited by divalent cations (K for Ba block of spermidine efflux is 0.1 mM), but not by tetraethylammonium ions or verapamil. Spermidine efflux rates were not different between control oocytes
and those expressing HRK1 inward rectifier K (Kir) channels. When the membrane potential was clamped, either by changing external [K] in oocytes expressing HRK1, or by 2-microelectrode voltage-clamp, spermidine efflux was shown to be strongly dependent on
voltage, as expected for a simple electrodiffusive process, where spermidine is the effluxing species. This result argues against spermidine diffusing out as an uncharged species, or in exchange for
similarly charged counterions. These results are the first conclusive demonstration of a simple electrodiffusive pathway for
spermidine efflux from cells.
Journal of Biological Chemistry 02/1996; 271(7):3392-3397. · 4.77 Impact Factor
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Luis F. Lopez-Santiago,
Laurence S. Meadows,
Sara J. Ernst,
Chunling Chen,
Jyoti Dhar Malhotra,
Dyke P. McEwen,
Audrey Speelman,
Jeffrey L. Noebels,
Sebastian K.G. Maier, Anatoli N. Lopatin,
Lori L. Isom
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ABSTRACT: In neurons, voltage-gated sodium channel β subunits regulate the expression levels, subcellular localization, and electrophysiological properties of sodium channel α subunits. However, the contribution of β subunits to sodium channel function in heart is poorly understood. We examined the role of β1 in cardiac excitability using Scn1b null mice. Compared to wildtype mice, electrocardiograms recorded from Scn1b null mice displayed longer RR intervals and extended QTc intervals, both before and after autonomic block. In acutely dissociated ventricular myocytes, loss of β1 expression resulted in a ∼ 1.6-fold increase in both peak and persistent sodium current while channel gating and kinetics were unaffected. Nav1.5 expression increased in null myocytes ∼ 1.3-fold. Action potential recordings in acutely dissociated ventricular myocytes showed slowed repolarization, supporting the extended QTc interval. Immunostaining of individual myocytes or ventricular sections revealed no discernable alterations in the localization of sodium channel α or β subunits, ankyrinB, ankyrinG, N-cadherin, or connexin-43. Together, these results suggest that β1 is critical for normal cardiac excitability and loss of β1 may be associated with a long QT phenotype.
Journal of Molecular and Cellular Cardiology.
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[hide abstract]
ABSTRACT: The lack of pathological consequences of cardiac ATP-sensitive potassium channel (KATP) channel gene manipulation is in stark contrast to the effect of similar perturbations in the pancreatic β-cell. Because the pancreatic and cardiac channel share the same pore-forming subunit (Kir6.2), the different effects of genetic manipulation likely reflect, at least in part, the tissue-specific expression of the regulatory subunit (SUR1 in pancreas vs. SUR2A in heart) of the bipartite channel complex. To examine this, we have generated transgenic (TG) mice that overexpress epitope-tagged SUR1 or SUR2A under the transcriptional control of the α-myosin heavy chain promoter. Western blot and real time RT-PCR analysis confirm transgene expression in the heart, and variable levels of SUR1 RNA and protein, in 16 viable founder lines. Surprisingly, activation of channels by either pharmacological agents (diazoxide and pinacidil) or metabolic inhibitors (oligomycin and 2-deoxyglucose) reveals a suppression of total KATP conductance in high expressing TG mice. Moreover, KATP channel activity was significantly reduced in excised cardiac patches from TG myocytes that overexpress either SUR1 or SUR2A. Using a recombinant cell system, we show that overexpression of either SUR1 or Kir6.2 suppresses the functional expression of KATP from optimized dimeric SUR1-Kir6.2. Thus, the graded effect of SUR1 expression in the intact heart appears to demonstrate an in vivo requirement for 1:1 expression ratio of Kir6.2 and SURx.
Journal of Molecular and Cellular Cardiology.
