Overexpression of a human potassium channel suppresses cardiac hyperexcitability in rabbit ventricular myocytes.

The high incidence of sudden death in heart failure may reflect abnormalities of repolarization and heightened susceptibility to arrhythmogenic early afterdepolarizations (EADs). We hypothesized that overexpression of the human K+ channel HERG (human ether-a-go-go-related gene) could enhance repolarization and suppress EADs. Adult rabbit ventricular myocytes were maintained in primary culture, which suffices to prolong action potentials and predisposes to EADs. To achieve efficient gene transfer, we created AdHERG, a recombinant adenovirus containing the HERG gene driven by a Rous sarcoma virus (RSV) promoter. The virally expressed HERG current exhibited pharmacologic and kinetic properties like those of native IKr. Transient outward currents in AdHERG-infected myocytes were similar in magnitude to those in control cells, while stimulated action potentials (0.2 Hz, 37 degrees C) were abbreviated compared with controls. The occurrence of EADs during a train of action potentials was reduced by more than fourfold, and the relative refractory period was increased in AdHERG-infected myocytes compared with control cells. Gene transfer of delayed rectifier potassium channels represents a novel and effective strategy to suppress arrhythmias caused by unstable repolarization.

A revised view of cardiac sodium channel "blockade" in the long-QT syndrome.

Mutations in SCN5A, encoding the cardiac sodium (Na) channel, are linked to a form of the congenital long-QT syndrome (LQT3) that provokes lethal ventricular arrhythmias. These autosomal dominant mutations disrupt Na channel function, inhibiting channel inactivation, thereby causing a sustained ionic current that delays cardiac repolarization. Sodium channel-blocking antiarrhythmics, such as lidocaine, potently inhibit this pathologic Na current (I(Na)) and are being evaluated in patients with LQT3. The mechanism underlying this effect is unknown, although high-affinity "block" of the open Na channel pore has been proposed. Here we report that a recently identified LQT3 mutation (R1623Q) imparts unusual lidocaine sensitivity to the Na channel that is attributable to its altered functional behavior. Studies of lidocaine on individual R1623Q single-channel openings indicate that the open-time distribution is not changed, indicating the drug does not block the open pore as proposed previously. Rather, the mutant channels have a propensity to inactivate without ever opening ("closed-state inactivation"), and lidocaine augments this gating behavior. An allosteric gating model incorporating closed-state inactivation recapitulates the effects of lidocaine on pathologic I(Na). These findings explain the unusual drug sensitivity of R1623Q and provide a general and unanticipated mechanism for understanding how Na channel-blocking agents may suppress the pathologic, sustained Na current induced by LQT3 mutations.

Adenovirus-mediated expression of a voltage-gated potassium channel in vitro (rat cardiac myocytes) and in vivo (rat liver). A novel strategy for modifying excitability.

Excitability is governed primarily by the complement of ion channels in the cell membrane that shape the contour of the action potential. To modify excitability by gene transfer, we created a recombinant adenovirus designed to overexpress a Drosophila Shaker potassium channel (AdShK). In vitro, a variety of mammalian cell types infected with AdShK demonstrated robust expression of the exogenous channel. Spontaneous action potentials recorded from cardiac myocytes in primary culture were abbreviated compared with noninfected myocytes. Intravascular infusion of AdShK in neonatal rats induced Shaker potassium channel mRNA expression in the liver, and large potassium currents could be recorded from explanted hepatocytes. Thus, recombinant adenovirus technology has been used for in vitro and in vivo gene transfer of ion channel genes designed to modify cellular action potentials. With appropriate targeting, such a strategy may be useful in gene therapy of arrhythmias, seizure disorders, and myotonic muscle diseases.

Local anesthetics as effectors of allosteric gating. Lidocaine effects on inactivation-deficient rat skeletal muscle Na channels.

Time- and voltage-dependent local anesthetic effects on sodium (Na) currents are generally interpreted using modulated receptor models that require formation of drug-associated nonconducting states with high affinity for the inactivated channel. The availability of inactivation-deficient Na channels has enabled us to test this traditional view of the drug-channel interaction. Rat skeletal muscle Na channels were mutated in the III-IV linker to disable fast inactivation (F1304Q: FQ). Lidocaine accelerated the decay of whole-cell FQ currents in Xenopus oocytes, reestablishing the wild-type phenotype; peak inward current at -20 mV was blocked with an IC50 of 513 microM, while plateau current was blocked with an IC50 of only 74 microM (P < 0.005 vs. peak). In single-channel experiments, mean open time was unaltered and unitary current was only reduced at higher drug concentrations, suggesting that open-channel block does not explain the effect of lidocaine on FQ plateau current. We considered a simple model in which lidocaine reduced the free energy for inactivation, causing altered coupling between activation and inactivation. This model readily simulated macroscopic Na current kinetics over a range of lidocaine concentrations. Traditional modulated receptor models which did not modify coupling between gating processes could not reproduce the effects of lidocaine with rate constants constrained by single-channel data. Our results support a reinterpretation of local anesthetic action whereby lidocaine functions as an allosteric effector to enhance Na channel inactivation.

Molecular interactions between two long-QT syndrome gene products, HERG and KCNE2, rationalized by in vitro and in silico analysis.

The cardiac delayed rectifier potassium current mediates repolarization of the action potential and underlies the QT interval of the ECG. Mutations in either of the two molecular components of the rapid delayed rectifier (I(K,r)), HERG and KCNE2, have been linked to heritable or acquired long-QT syndrome. Mechanisms whereby mutations of KCNE2 produce fatal cardiac arrhythmias characteristic of long-QT syndrome remain unclear. In this study, we characterize functional interactions between HERG and KCNE2 with a view to defining underlying mechanisms for action potential prolongation and long-QT syndrome. Whereas coexpression of hKCNE2 with HERG alters both kinetics and density of ionic current, incorporation of these effects into a quantitative model of the action potential predicts that only changes in current density significantly affect repolarization. Thus, the primary functional consequence of hKCNE2 on action potential morphology is through modulation of I(K,r) density, as predicted by the model. Mutations associated with long-QT syndrome that result only in modest changes of gating kinetics may be epiphenomena or may modulate action potential repolarization via interaction with alternative pore-forming potassium channel alpha subunits.

Cardiac sodium channels (hH1) are intrinsically more sensitive to block by lidocaine than are skeletal muscle (mu 1) channels.

When lidocaine is given systemically, cardiac Na channels are blocked preferentially over those in skeletal muscle and nerve. This apparent increased affinity is commonly assumed to arise solely from the fact that cardiac Na channels spend a large fraction of their time in the inactivated state, which exhibits a high affinity for local anesthetics. The oocyte expression system was used to compare systematically the sensitivities of skeletal (mu 1-beta 1) and cardiac (hH1-beta 1) Na channels to block by lidocaine, under conditions in which the only difference was the choice of alpha subunit. To check for differences in tonic block, Na currents were elicited after 3 min of exposure to various lidocaine concentrations at -100 mV, a potential at which both hH1-beta 1 and mu 1-beta 1 channels were fully reprimed. Surprisingly, hH1-beta 1 Na channels were threefold more sensitive to rested-state block by lidocaine (402 +/- 36 microM, n = 4-22) than were mu 1-beta 1 Na channels (1,168 +/- 34 microM, n = 7-19). In contrast, the inactivated state binding affinities determined at partially depolarized holding potentials (h infinity approximately 0.2) were similar (Kd = 16 +/- 1 microM, n = 3-9 for hH1-beta 1 and 12 +/- 2 microM, n = 4-11 for mu 1-beta 1). Lidocaine produced more use-dependent block of peak hH1-beta 1 Na current elicited by trains of short-(10 ms) or long- (1 s) duration step depolarizations (0.5 Hz, -20 mV) than of mu 1-beta 1 Na current. During exposure to lidocaine, hH1-beta 1 channels recover from inactivation at -100 mV after a prolonged delay (20 ms), while mu 1-beta 1 channels begin repriming immediately. The overall time course of recovery from inactivation in the presence of lidocaine is much slower in hH1-beta 1 than in mu 1-beta 1 channels. These unexpected findings suggest that structural differences in the alpha subunits impart intrinsically different lidocaine sensitivities to the two isoforms. The differences in steady state affinities and in repriming kinetics are both in the correct direction to help explain the increased potency of cardiac Na channel block by local anesthetics.

Functional consequences of lidocaine binding to slow-inactivated sodium channels.

Na channels open upon depolarization but then enter inactivated states from which they cannot readily reopen. After brief depolarizations, native channels enter a fast-inactivated state from which recovery at hyperpolarized potentials is rapid (< 20 ms). Prolonged depolarization induces a slow-inactivated state that requires much longer periods for recovery (> 1 s). The slow-inactivated state therefore assumes particular importance in pathological conditions, such as ischemia, in which tissues are depolarized for prolonged periods. While use-dependent block of Na channels by local anesthetics has been explained on the basis of delayed recovery of fast-inactivated Na channels, the potential contribution of slow-inactivated channels has been ignored. The principal (alpha) subunits from skeletal muscle or brain Na channels display anomalous gating behavior when expressed in Xenopus oocytes, with a high percentage entering slow-inactivated states after brief depolarizations. This enhanced slow inactivation is eliminated by coexpressing the alpha subunit with the subsidiary beta 1 subunit. We compared the lidocaine sensitivity of alpha subunits expressed in the presence and absence of the beta 1 subunit to determine the relative contributions of fast-inactivated and slow-inactivated channel block. Coexpression of beta 1 inhibited the use-dependent accumulation of lidocaine block during repetitive (1-Hz) depolarizations from -100 to -20 mV. Therefore, the time required for recovery from inactivated channel block was measured at -100 mV. Fast-inactivated (alpha + beta 1) channels were mostly unblocked within 1 s of repolarization; however, slow-inactivated (alpha alone) channels remained blocked for much longer repriming intervals (> 5 s). The affinity of the slow-inactivated state for lidocaine was estimated to be 15-25 microM, versus 24 microM for the fast-inactivated state. We conclude that slow-inactivated Na channels are blocked by lidocaine with an affinity comparable to that of fast-inactivated channels. A prominent functional consequence is potentiation of use-dependent block through a delay in repriming of lidocaine-bound slow-inactivated channels.

Functional association of the beta 1 subunit with human cardiac (hH1) and rat skeletal muscle (mu 1) sodium channel alpha subunits expressed in Xenopus oocytes.

Native cardiac and skeletal muscle Na channels are complexes of alpha and beta 1 subunits. While structural correlates for activation, inactivation, and permeation have been identified in the alpha subunit and the expression of alpha alone produces functional channels, beta 1-deficient rat skeletal muscle (mu 1) and brain Na channels expressed in Xenopus oocytes do not gate normally. In contrast, the requirement of a beta 1 subunit for normal function of Na channels cloned from rat heart or human heart (hH1) has been disputed. Coinjection of rat brain beta 1 subunit cRNA with hH1 (or mu 1) alpha subunit cRNA into oocytes increased peak Na currents recorded 2 d after injection by 240% (225%) without altering the voltage dependence of activation. In mu 1 channels, steady state inactivation was shifted to more negative potentials (by 6 mV, p < 0.01), but the shift of 2 mV was not significant for hH1 channels. Nevertheless, coexpression with beta 1 subunit speeded the decay of macroscopic current of both isoforms. Ensemble average hH1 currents from cell-attached patches revealed that coexpression of beta 1 increases the rate of inactivation (quantified by time to 75% decay of current; p < 0.01 at -30, -40, and -50 mV). Use-dependent decay of hH1 Na current during repeated pulsing to -20 mV (1 s, 0.5 Hz) after a long rest was reduced to 16 +/- 2% of the first pulse current in oocytes coexpressing alpha and beta 1 subunits compared to 35 +/- 8% use-dependent decay for oocytes expressing the alpha subunit alone. Recovery from inactivation of mu 1 and hH1 Na currents after 1-s pulses to -20 mV is multiexponential with three time constants; coexpression of beta 1 subunit decreased all three recovery time constants. We conclude that the beta 1 subunit importantly influences the function of Na channels produced by coexpression with either the hH1 or mu 1 alpha subunits.

Repolarization abnormalities, arrhythmia and sudden death in canine tachycardia-induced cardiomyopathy.

OBJECTIVES: This study sought to determine whether the canine model of tachycardia-induced heart failure (HF) is an effective model for sudden cardiac death (SCD) in HF. BACKGROUND: Such a well established HF model that also exhibits arrhythmias and SCD, along with repolarization abnormalities that could trigger them, may facilitate the study of SCD in HF, which still eludes effective treatment. METHODS: Twenty-five dogs were VVI-paced at 250 beats/min for 3 to 5 weeks. Electrocardiograms were obtained, and left ventricular endocardial monophasic action potentials (MAPs) were recorded at six sites at baseline and after HF. Weekly Holter recordings were made with pacing suspended for 24 h. RESULTS: Six animals (24%) died suddenly, one with Holter-documented polymorphic ventricular tachycardia (VT). Holter recordings revealed an increased incidence of VT as HF progressed. Repolarization was significantly (p < 0.05) prolonged, as indexed by a corrected QT interval (mean [+/-SD] 311 +/- 25 to 338 +/- 25 ms) and MAP duration measured at 90% repolarization (MAPD90) (181 +/- 19 to 209 +/- 28 ms), and spatial MAPD90 dispersion rose by 40%. We further tested whether CsCl inhibition of repolarizing K+ currents, which are reportedly downregulated in HF, might preferentially prolong the MAPD90 in HF. With 1 mEq/kg body weight of CsCl, MAPD90 rose by 86 +/- 100 ms in dogs with HF versus only 28 +/- 16 ms in control animals (p = 0.002). Similar disparities in CsCl sensitivity were observed in myocytes isolated from normal and failing hearts. CONCLUSIONS: Tachycardia-induced HF exhibits malignant arrhythmia and SCD, along with prolonged, heterogeneous repolarization and heightened sensitivity to CsCl at chamber and cellular levels. Thus, it appears to be a useful model for studying mechanisms and therapy of SCD in HF.

Effect of duration of depolarisation on contraction of normal and hypertrophied feline ventricular myocytes.

OBJECTIVE: The aim was to test the hypothesis that the prolongation of action potential duration in hypertrophied feline myocytes causes the contractions to be of long duration. METHODS: Left ventricular hypertrophy was induced by slow progressive pressure overload after banding the ascending aorta of young cats. Single myocytes were enzymatically dissociated for whole cell patch clamp studies. Cell shortenings were induced by stimulated action potentials (in current clamp mode) and by step depolarisations using voltage clamp to control the duration of depolarisation. RESULTS: Action potential duration measured at 50% repolarisation (0.5 Hz) was significantly longer in hypertrophied myocytes, at 688(SEM 43) ms, n = 25, v 396(17) ms, n = 22, in control myocytes (p < 0.01). The associated contractions in hypertrophied myocytes had significantly longer durations measured at 50% relengthening [hypertrophied myocytes 609(54) ms, control myocytes 406(13) ms]. The absolute magnitude of shortening normalised to percent diastolic cell length was also significantly reduced in hypertrophied myocytes [7.8(0.8)% diastolic cell length] compared to control myocytes [12.2(0.6)% diastolic cell length] and the duration of contraction time to 50% relengthening was prolonged [406(13) ms v 609(54) ms]. When the duration of depolarisation was controlled with voltage clamp techniques, steady state contractions at +10 mV increased in magnitude in both control and hypertrophied myocytes as the duration of depolarization was lengthened. At all durations tested (100-1000 ms), contractions were significantly longer in duration in hypertrophied myocytes. Changing the duration of depolarisation had no significant effect on the duration of contraction in control myocytes. In hypertrophied myocytes, however, prolongation of depolarisation (500-1000 ms) significantly prolonged the contraction. Steady state contractions initiated from -70 mV (sodium current activated) were larger in both control and hypertrophied myocytes than contractions elicited from -40 mV (sodium current inactivated), and the effect of depolarisation duration on contractile duration was the same. CONCLUSIONS: Changes in sarcolemmal properties which produce a lengthening of the action potential duration in hypertrophy are not primarily responsible for the prolongation of contractile duration. However, there is a portion of contraction which becomes sensitive to the duration of depolarisation in hypertrophied myocytes.

Prospects for genetic manipulation of cardiac excitability.

Despite impressive advances in the therapy of a number of types of heart disease in the last two decades, sudden cardiac death remains a public health problem of staggering dimensions. Current treatment options include antiarrhythmic drugs that have higher than desired failure rates and implantable defibrillators that incur significant costs to the patient and society. The development of therapies that better suppress the cardiac arrhythmias responsible for sudden cardiac death requires a broad and comprehensive understanding of the basic mechanisms underlying electrical instability in the heart. This study explores the scientific basis for a molecular genetic approach to modify cardiac excitability and thereby to create animal models of sudden cardiac death. The availability of such models will open up new avenues of research in arrhythmogenesis and facilitate the development of novel antiarrhythmic agents.

Sodium-calcium exchange-mediated contractions in feline ventricular myocytes.

The hypothesis that Ca entry by the sarcolemmal Na-Ca exchange mechanism induces sarcoplasmic reticulum (SR) Ca release, loads the SR with Ca, and/or directly induces contractions by elevating cytosolic free Ca was tested in voltage-clamped feline ventricular myocytes. Intracellular Na concentration was increased by cellular dialysis to enhance Ca influx via "reverse-mode" Na-Ca exchange at positive membrane potentials, at which the "L-type" Ca current (ICa) should be small. Contractions were induced in the presence of Ca channel antagonists by depolarization to these potentials, suggesting that Ca influx via reverse-mode Na-Ca exchange was involved. These contractions had both phasic (SR related) and tonic components of shortening. They were smaller and began with more delay after depolarization than contractions which involved ICa. The magnitude of shortening was graded by the amount and duration of depolarization, suggesting that Ca influx via reverse-mode Na-Ca exchange has the capacity to induce and grade SR Ca release. Small slow contractions could be evoked in the presence of ryanodine (to impair SR function) and verapamil (to block ICa), supporting the idea that Ca influx via Na-Ca exchange is sufficient to directly activate the contractile proteins. Contractions induced by voltage steps to +10 mV, which were usually small when ICa was blocked, were potentiated if preceded by a voltage step to strongly positive potentials. This potentiation was inhibited by ryanodine, suggesting that Ca entry that occurs by Na-Ca exchange may be important for normal SR Ca loading.(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage dependence of contraction and calcium current in severely hypertrophied feline ventricular myocytes.

In the present study the voltage dependence of contraction and the characteristics of the "L-type" Ca2+ current (ICa) were compared in control (C) and severely hypertrophied (H) myocytes (M). Hypertrophy was induced in young cats by slow progressive pressure overload of the feline right ventricle. The amount of hypertrophy induced in this study was more severe than previously examined in this laboratory. The major findings of this study were: (1) The voltage dependence of contraction was not significantly different in C and HM. Peak shortening occurred at 6.2 +/- 1.2 mV in HM and at 9.5 +/- 1.3 mV in CM. (2) The magnitude of peak ICa density was significantly smaller in HM (5.56 +/- 0.53 pA/pF; n = 17) than in Cm (7.09 +/- 0.42 pA/pF; n = 20). In both groups of cells ICa reached a maximum at + 10 mV. (3) There were no significant changes in the voltage dependence of both ICa activation and steady-state inactivation. This is the first study to provide evidence that ICa density is reduced in severe hypertrophy. The 21% decrease in peak ICa density could reduce the contractile state of the heart if calcium-induced calcium release is normal and the reduction of ICa alters the Ca2+ released from the sarcoplasmic reticulum. The reduction in "L-type" Ca2+ current density in severely hypertrophied myocytes may play a role in the transition from the compensated hypertrophic state to congestive heart failure. Data are means +/- standard error.

T-type Ca2+ current is expressed in hypertrophied adult feline left ventricular myocytes.

Macroscopic T-type Ca2+ currents, which are often observed in fetal and neonatal cardiac muscle cells, were not found in normal (0 of 17) adult feline ventricular myocytes. However, they were present in most (15 of 21) myocytes isolated from adult feline left ventricles with long-standing pressure-overload-induced hypertrophy. This is the first study to provide evidence in a large mammal, such as the cat, that T-type Ca2+ channels may be reexpressed in adults in association with hypertrophy resulting from slow progressive pressure overload. Importantly, this expression was stable for the duration of the hypertrophy process and was not associated with abrupt pressure overload. T-type Ca2+ currents were separated from L-type Ca2+ currents by exploiting the differences in their voltage dependence of steady-state inactivation. Depolarizations from -80 mV revealed a rapidly activating inward current that peaked in magnitude at -30 mV (-1.8 +/- 0.9 [mean +/- SD] pA/pF) and fully inactivated within 100 milliseconds in 15 of 21 hypertrophied myocytes studied. Further depolarizations activated progressively less T-type Ca2+ current, so that at +10 mV the L-type Ca2+ current predominated. In the hypertrophied myocytes that demonstrated both T-type and L-type Ca2+ currents, two distinct peaks occurred in their current-voltage relations. T-type Ca2+ currents were not evident in any of the 17 normal adult feline left ventricular myocytes studied. The purpose of T-type Ca2+ currents in hypertrophy is unclear. However, their presence may make hypertrophied myocardium more prone to spontaneous action potentials and increase the likelihood for arrhythmias in partially depolarized hypertrophied myocardium.

Electrophysiological properties of neonatal mouse cardiac myocytes in primary culture.

1. The increasing utility of transgenic mice in molecular studies of the cardiovascular system has motivated us to characterize the ionic currents in neonatal mouse ventricular myocytes. 2. Cell capacitance measurements (30 +/- 1 pF, n = 73) confirmed visual impressions that neonatal mouse ventricular myocytes in primary culture are considerably smaller than freshly isolated adult ventricular myocytes. With the use of electron microscopy, mitochondria and sarcoplasmic reticulum were found in close association with myofibrils, but transverse tubules were not observed. 3. Action potential durations, measured at 50 and 90% repolarization, were 23 +/- 1 and 42 +/- 2 ms respectively (n = 46). Application of 4-aminopyridine (4-AP; 5 mM) prolonged action potential duration at 50% repolarization by 26 +/- 5% (n = 3). The brevity of the action potential is explained by the rapid activation of a transient outward K+ current upon voltage-clamp depolarization to plateau potentials. 4. Potassium currents identified include an inward rectifier, a large 4-AP-sensitive transient outward, a slowly inactivating 4-AP-insensitive outward, a slowly activating delayed rectifier and a small rapidly activating E-4031 (10 microM)-sensitive delayed rectifier K+ current. 5. Sodium currents (-305 +/- 50 pA pF-1, n = 21) were recorded in 40 mM Na+ with Ni2+ (1 mM) to block Ca2+ currents and with K+ replaced by Cs+. The relative insensitivity of the Na+ current to block by tetrodotoxin (IC50 = 2.2 +/- 0.3 microM, n = 4) is distinctive of the cardiac Na+ channel isoform. 6. Nitrendipine-insensitive (10 microM) Ba2+ currents elicited during steps from -90 to -30 mV measured -25 +/- 5 pA pF-1 (n = 7, 30 mM Ba2+). Decay of these currents was complete during 180 ms depolarizations, even with Ba2+ as the charge carrier. These currents were not present when the holding potential was set at -50 mV. These data support the presence of a low threshold, T-type Ca2+ current. 7. The maximal nitrendipine-sensitive L-type Ca2+ current density was -10 +/- 2 pA pF-1 (n = 8) in 2 mM Ca2+ and -38 +/- 5 pA pF-1 (n = 9) in 30 mM Ba2+. Exposure to isoprenaline (1 microM) resulted in an 82% increase (n = 3) in the amplitude of the Ba2+ currents elicited at 0 mV. 8. Neonatal mouse cardiac myocytes in primary culture possess surprisingly large inward currents given the brevity of their action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of neuronal and cardiac transient outward currents by viral gene transfer of dominant-negative Kv4.2 constructs.

To probe the molecular identity of transient outward (A-type) potassium currents, we expressed a truncated version of Kv4.2 in heart cells and neurons. The rat Kv4.2-coding sequence was truncated at a position just past the first transmembrane segment and subcloned into an adenoviral shuttle vector downstream of a cytomegalovirus promoter (pE1Kv4.2ST). We hypothesized that this construct would act as a dominant-negative suppressor of currents encoded by the Kv4 family by analogy to Kv1 channels. Cotransfection of wild-type Kv4.2 with a beta-galactosidase expression vector in Chinese hamster ovary (CHO)-K1 cells produced robust transient outward currents (Ito) after two days (14.0 pA/pF at 50 mV, n = 5). Cotransfection with pE1Kv4.2ST markedly suppressed the Kv4.2 currents (0.8 pA/pF, n = 6, p < 0.02; cDNA ratio of 2:1 Kv4.2ST:wild type), but in parallel experiments, it did not alter the current density of coexpressed Kv1.4 or Kv1.5 channels. Kv4.2ST also effectively suppressed rat Kv4.3 current when coexpressed in CHO-K1 cells. We then engineered a recombinant adenovirus (AdKv4.2ST) designed to overexpress Kv4.2ST in infected cells. A-type currents in rat cerebellar granule cells were decreased two days after AdKv4. 2ST infection as compared with those infected by a beta-galactosidase reporter virus (116.0 pA/pF versus 281.4 pA/pF in Ad beta-galactosidase cells, n = 8 each group, p < 0.001). Likewise, Ito in adult rat ventricular myocytes was suppressed by AdKv4.2ST but not by Adbeta-galactosidase (8.8 pA/pF versus 21.4 pA/pF in beta-galactosidase cells, n = 6 each group, p < 0.05). Expression of a GFP-Kv4.2ST fusion construct enabled imaging of subcellular protein localization by confocal microscopy. The protein was distributed throughout the surface membrane and intracellular membrane systems. We conclude that genes from the Kv4 family are the predominant contributors to the A-type currents in cerebellar granule cells and Ito in rat ventricle. Overexpression of dominant-negative constructs may be of general utility in dissecting the contributions of various ion channel genes to excitability.

Somatic gene transfer of tagged K+ channel fragments to probe trafficking and electrical function in epithelial cells and cardiac myocytes.

To evaluate the roles of the C-termini of K + channels in subcellular targeting and protein-protein interactions, we created fusion constructs of the cell-surface antigen CD8 and the C-termini of Kv4.3, Kv1.4 and KvLQT1. Using a Cre-lox recombination system, we made 3 adenoviruses containing a fusion of the N-terminal-and transmembrane segments of CD8 with the C-termini of each of the 3 K + channels. Expression in polarized Opossum Kidney (OK) epithelial cells led to localization of CD8-Kv4.3 and CD8-Kv1.4 into the apical and basolateral membranes, while CD8-KvLQT1 remained in the endoplasmic reticulum (ER), even when co-expressed with MinK. When expressed in rat cardiac myocytes in culture, all the 3 constructs were diffusely targeted to the surface membrane. The ER retention of CD8-KvLQT1 in OK cells but not in cardiomyocytes thus reveals functional differences in trafficking between these two cell types. To probe functional roles of C-termini, we studied K + currents in cardiac myocytes expressing CD8-Kv4.3. Patch-clamp recordings of transient outward current revealed a hyperpolarizing shift of steady-state inactivation, implying that CD8-Kv4.3 may be disrupting the interaction of Kv4.x channels with one or more as-yet-undefined regulatory subunits. Thus, expression of tagged ion-channel fragments represents a novel, generalizable approach that may help to elucidate assembly, localization and function of these important signaling proteins.

Cellular basis of ventricular arrhythmias and abnormal automaticity in heart failure.

The high incidence of sudden death in heart failure may reflect an increased propensity to abnormal repolarization and long Q-T interval-related arrhythmias. If so, cells from failing hearts would logically be expected to exhibit a heightened susceptibility to early afterdepolarizations (EAD). We found that midmyocardial ventricular cells isolated from dogs with pacing-induced heart failure exhibited an increased action potential duration and many more EAD than cells from nonpaced controls; this was the case both under basal conditions (P < 0.01) and after lowering external K(+) concentration ([K(+)](o)) to 2 mM and exposing cells to cesium (3 mM; P < 0.05). An unexpected finding was the occurrence of spontaneous depolarizations (SD, >5 mV) from the resting potential that were not coupled to prior action potentials. These SD were observed in 20% of failing cells (n = 5 of 25) under basal ionic conditions but in none of the normal cells (n = 0 of 27, P < 0.05). The net inward current that underlies SD is not triggered by Ca(2+) oscillations and thus differs fundamentally from the currents that underlie delayed afterdepolarizations. We conclude that cardiomyopathic canine ventricular cells are intrinsically predisposed to EAD and SD. Because EAD have been linked to the pathogenesis of torsade de pointes, our results support the hypothesis that sudden death in heart failure often arises from abnormalities of repolarization. The frequent occurrence of SD points to a novel cellular mechanism for abnormal automaticity in heart failure.

Isoform-specific lidocaine block of sodium channels explained by differences in gating.

When depolarized from typical resting membrane potentials (V(rest) approximately -90 mV), cardiac sodium (Na) currents are more sensitive to local anesthetics than brain or skeletal muscle Na currents. When expressed in Xenopus oocytes, lidocaine block of hH1 (human cardiac) Na current greatly exceeded that of mu1 (rat skeletal muscle) at membrane potentials near V(rest), whereas hyperpolarization to -140 mV equalized block of the two isoforms. Because the isoform-specific tonic block roughly parallels the drug-free voltage dependence of channel availability, isoform differences in the voltage dependence of fast inactivation could underlie the differences in block. However, after a brief (50 ms) depolarizing pulse, recovery from lidocaine block is similar for the two isoforms despite marked kinetic differences in drug-free recovery, suggesting that differences in fast inactivation cannot entirely explain the isoform difference in lidocaine action. Given the strong coupling between fast inactivation and other gating processes linked to depolarization (activation, slow inactivation), we considered the possibility that isoform differences in lidocaine block are explained by differences in these other gating processes. In whole-cell recordings from HEK-293 cells, the voltage dependence of hH1 current activation was approximately 20 mV more negative than that of mu1. Because activation and closed-state inactivation are positively coupled, these differences in activation were sufficient to shift hH1 availability to more negative membrane potentials. A mutant channel with enhanced closed-state inactivation gating (mu1-R1441C) exhibited increased lidocaine sensitivity, emphasizing the importance of closed-state inactivation in lidocaine action. Moreover, when the depolarization was prolonged to 1 s, recovery from a "slow" inactivated state with intermediate kinetics (I(M)) was fourfold longer in hH1 than in mu1, and recovery from lidocaine block in hH1 was similarly delayed relative to mu1. We propose that gating processes coupled to fast inactivation (activation and slow inactivation) are the key determinants of isoform-specific local anesthetic action.