Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature.

We have established stably transfected HEK 293 cell lines expressing high levels of functional human ether-a go-go-related gene (HERG) channels. We used these cells to study biochemical characteristics of HERG protein, and to study electrophysiological and pharmacological properties of HERG channel current at 35 degrees C. HERG-transfected cells expressed an mRNA band at 4.0 kb. Western blot analysis showed two protein bands (155 and 135 kDa) slightly larger than the predicted molecular mass (127 kDa). Treatment with N-glycosidase F converted both bands to smaller molecular mass, suggesting that both are glycosylated, but at different levels. HERG current activated at voltages positive to -50 mV, maximum current was reached with depolarizing steps to -10 mV, and the current amplitude declined at more positive voltages, similar to HERG channel current expressed in other heterologous systems. Current density at 35 degrees C, compared with 23 degrees C, was increased by more than twofold to a maximum of 53.4 +/- 6.5 pA/pF. Activation, inactivation, recovery from inactivation, and deactivation kinetics were rapid at 35 degrees C, and more closely resemble values reported for the rapidly activating delayed rectifier K+ current (I(Kr)) at physiological temperatures. HERG channels were highly selective for K+. When we used an action potential clamp technique, HERG current activation began shortly after the upstroke of the action potential waveform. HERG current increased during repolarization to reach a maximum amplitude during phases 2 and 3 of the cardiac action potential. HERG contributed current throughout the return of the membrane to the resting potential, and deactivation of HERG current could participate in phase 4 depolarization. HERG current was blocked by low concentrations of E-4031 (IC50 7.7 nM), a value close to that reported for I(Kr) in native cardiac myocytes. Our data support the postulate that HERG encodes a major constituent of I(Kr) and suggest that at physiological temperatures HERG contributes current throughout most of the action potential and into the postrepolarization period.     

Kv11.1 (ERG1) K+ channels localize in cholesterol and sphingolipid enriched membranes and are modulated by membrane cholesterol

The localization of ion channels to specific membrane microdomains can impact the functional properties of channels and their role in cellular physiology. We determined the membrane localization of human Kv11.1 (hERG1) alpha-subunit protein, which underlies the rapidly activating, delayed rectifier K(+) current (I(Kr)) in the heart. Immunocytochemistry and membrane fractionation using discontinuous sucrose density gradients of adult canine ventricular tissue showed that Kv11.1 channel protein localized to both the cell surface and T-tubular sarcolemma. Furthermore, density gradient membrane fractionation using detergent (Triton X-100) and non-detergent (OptiPrep) methods from canine ventricular myocytes or HEK293 cells demonstrated that Kv11.1 protein, along with MiRP1 and Kv7.1 (KCNQ1) proteins, localize in cholesterol and sphingolipid enriched membrane fractions. In HEK293 cells, Kv11.1 channels, but not long QT-associated mutant G601S-Kv11.1 channels, also localized to cholesterol and sphingolipid enriched membrane fractions. Depletion of membrane cholesterol from HEK293 cells expressing Kv11.1 channels using methyl-beta-cyclodextrin (MbetaCD) caused a positive shift of the voltage dependence of activation and an acceleration of deactivation kinetics of Kv11.1 current (I(Kv11.1)). Cholesterol loading of HEK293 cells reduced the steep voltage dependence of I(Kv11.1) activation and accelerated the inactivation kinetics of I(Kv11.1). Incubation of neonatal mouse myocytes in MbetaCD also accelerated the deactivation kinetics of I(Kr). We conclude that Kv11.1 protein localizes in cholesterol and sphingolipid enriched membranes and that membrane cholesterol can modulate I(Kv11.1) and I(Kr).     

L-type Ca2+ currents in ventricular myocytes from neonatal and adult rats

Postnatal changes in the slow Ca2+ current (I(Ca)(L)) were investigated in freshly isolated ventricular myocytes from neonatal (1-7 days old) and adult (2-4 months old) rats, using whole-cell voltage clamp and single-channel recordings. The membrane capacitance (mean+/-SEM) averaged 23.2+/-0.5 pF in neonates (n = 163) and 140+/-4.1 pF in adults (n = 143). I(Ca)(L) was measured as the peak inward current at a test potential of +10 mV (or +20 mV) by applying a 300-ms pulse from a holding potential of -40 mV; 1.8 mM Ca2+ was used as charge carrier. The basal ICa(L) density was 6.7+/-0.2 pA/pF in neonatal and 7.8+/-0.2 pA/pF in adult cells (p < 0.05). The time course of inactivation of the fast component (at +10 ms) was significantly longer in the neonatal (10.7+/-1.4 ms) than in the adult (6.6+/-0.4 ms) cells (p < 0.05). Ryanodine (10+/-M) significantly increased this value to 18.0+/-1.9 in neonate (n = 8) and to 17.7+/-2.0 in adult (n = 9). For steady-state inactivation, the half-inactivation potential (Vh) was not changed in either group. For steady-state activation, Vh was 5.1 mV in the neonatal (n = 6) and -7.9 mV in the adult cells (n = 7). Single-channel recordings revealed that long openings (mode-2 behavior) were occasionally observed in the neonatal cells (11 events from 1080 traces/11 cells), but not in the adult cells (400 traces/4 cells). Slope conductance was 24 pS in both the neonatal and adult cells. Results in rat ventricular myocytes suggest the following: (i) the peak Ca2+ current density is already well developed in the neonatal period (being about 85% of the adult value); (ii) the fast component of inactivation is slower in neonates than in adults; and (iii) naturally occurring long openings are occasionally observed in the neonatal stage but not in the adult. Thus, the L-type Ca2+ channels of the neonate were slightly lower in density, were inactivated more slowly, and occasionally exhibited mode-2 behavior as compared with those of the adult.     

Characterization and functional consequences of delayed rectifier current transient in ventricular repolarization

Although inactivation of the rapidly activating delayed rectifier current (I(Kr)) limits outward current on depolarization, the role of I(Kr) (and recovery from inactivation) during repolarization is uncertain. To characterize I(Kr) during ventricular repolarization (and compare with the inward rectifier current, I(K1)), voltage-clamp waveforms simulating the action potential were applied to canine ventricular, atrial, and Purkinje myocytes. In ventricular myocytes, I(Kr) was minimal at plateau potentials but transiently increased during repolarizing ramps. The I(Kr) transient was unaffected by repolarization rate and maximal after 150-ms depolarizations (+25 mV). Action potential clamps revealed the I(Kr) transient terminating the plateau. Although peak I(Kr) transient density was relatively uniform among myocytes, potentials characterizing the peak transients were widely dispersed. In contrast, peak inward rectifier current (I(K1)) density during repolarization was dispersed, whereas potentials characterizing I(K1) defined a narrower (more negative) voltage range. In summary, rapidly activating I(Kr) provides a delayed voltage-dependent (and functionally time-independent) outward transient during ventricular repolarization, consistent with rapid recovery from inactivation. The heterogeneous voltage dependence of I(Kr) provides a novel means for modulating the contribution of this current during repolarization.     

Effects of divalent cations on the E-4031-sensitive repolarization current, I(Kr), in rabbit ventricular myocytes.

The effects of divalent cations on the E-4031-sensitive repolarization current (I(Kr)) were studied in single ventricular myocytes isolated from rabbit hearts. One group of divalent cations (Cd2+, Ni2+, Co2+, and Mn2+) produced a rightward shift of the I(Kr) activation curve along the voltage axis, increased the maximum I(Kr) amplitude (i.e., relieved the apparent inward rectification of the channel), and accelerated I(Kr) tail current kinetics. Another group (Ca2+, Mg2+ and Sr2+) had relatively little effect on I(Kr). The only divalent cation that blocked I(Kr) was Zn2+ (0.1-1 mM). Under steady-state conditions, Ba2+ caused a substantial block of I(K1) as previously reported. However, block by Ba2+ was time dependent, which precluded a study of Ba2+ effects on I(Kr). We conclude that the various effects of the divalent cations can be attributed to interactions with distinct sites associated with the rectification and/or inactivation mechanism of the channel.     

IKr and IKs remodeling differentially affects QT interval prolongation and dynamic adaptation to heart rate acceleration in bradycardic rabbits

Bradycardic ventricular electrical remodeling predisposes to lethal tachyarrhythmias. We investigated the early temporal sequence and reversibility of electrical remodeling in a rabbit complete heart block model subjected to bradycardic ventricular pacing for either 2 or 8 days, with a third group of animals undergoing 8 days of bradycardic pacing followed by 8 days of physiological-rate pacing. At specified time points after complete heart block induction and pacing initiation, steady-state QT interval measurements and variability as well as dynamic QT interval adaptation to abrupt heart rate acceleration were assessed in the absence and presence of isoproterenol. Rapidly (I(Kr)) and slowly (I(Ks)) activating delayed rectifier repolarizing K(+) tail current densities were evaluated using whole cell patch clamp in isolated right ventricular myocytes. Steady-state QT interval prolongation at both 2 and 8 days was associated with moderate I(Kr) reduction. I(Ks) downregulation was apparent by day 2 but more profound at day 8. Dynamic QT interval adaptation was impaired under baseline conditions at day 8 but only during isoproterenol administration at day 2. Both in vivo and cellular manifestations of remodeling reverted toward control values after 8 days of physiological-rate pacing. In conclusion, in this bradycardic model, I(Ks) downregulation 1) proceeds more gradually but more extensively than that of I(Kr) and 2) is most prominently associated with impaired dynamic QT interval adaptation to heart rate acceleration. Isoproterenol blunts the dynamic QT interval response in animals with partially downregulated I(Ks), consistent with stress-related phenomena in known I(Ks)-impaired states. Relative early sparing of I(Ks) could explain the delay in the onset of lethal tachyarrhythmia predisposition in bradycardic electrical remodeling. Reversibility of remodeling supports the potential utility of preventive pacing intervention soon after bradycardia onset.     

T-type calcium currents in rat cardiomyocytes during postnatal development: contribution to hormone secretion

T-type Ca(2+) channels have been suggested to play a role in cardiac automaticity, cell growth, and cardiovascular remodeling. Although three genes encoding for a T-type Ca(2+) channel have been identified, the nature of the isoform(s) supporting the cardiac T-type Ca(2+) current (I(Ca,T)) has not yet been determined. We describe the postnatal evolution of I(Ca,T) density in freshly dissociated rat atrial and ventricular myocytes and its functional properties at peak current density in young atrial myocytes. I(Ca,T) displays a classical low activation threshold, rapid inactivation kinetics, negative steady-state inactivation, slow deactivation, and the presence of a window current. Interestingly, I(Ca,T) is poorly sensitive to Ni(2+) and insensitive to R-type current toxin SNX-482. RT-PCR experiments and comparison of functional properties with recombinant Ca(2+) channel subtypes suggest that neonatal I(Ca,T) is related to the alpha(1G)-subunit. Atrial natriuretic factor (ANF) secretion was measured using peptide radioimmunoassays in atrial tissue. Pharmacological dissection of ANF secretion indicates an important contribution of I(Ca,T) to Ca(2+) signaling during the neonatal period.     

L-type but not T-type calcium current changes during postnatal development in rabbit sinoatrial node

Although the neonatal sinus node beats at a faster rate than the adult, when a sodium current (I(Na)) present in the newborn is blocked, the spontaneous rate is slower in neonatal myocytes than in adult myocytes. This suggests a possible functional substitution of I(Na) by another current during development. We used ruptured [T-type calcium current (I(Ca,T))] and perforated [L-type calcium current (I(Ca,L))] patch clamps to study developmental changes in calcium currents in sinus node cells from adult and newborn rabbits. I(Ca,T) density did not differ with age, and no significant differences were found in the voltage dependence of activation or inactivation. I(Ca,L) density was lower in the adult than newborn (12.1 +/- 1.4 vs. 17.6 +/- 2.5 pA/pF, P = 0.049). However, activation and inactivation midpoints were shifted in opposite directions, reducing the potential contribution during late diastolic depolarization in the newborn (activation midpoints -17.3 +/- 0.8 and -22.3 +/- 1.4 mV in the newborn and adult, respectively, P = 0.001; inactivation midpoints -33.4 +/- 1.4 and -28.3 +/- 1.7 mV for the newborn and adult, respectively, P = 0.038). Recovery of I(Ca,L) from inactivation was also slower in the newborn. The results suggest that a smaller but more negatively activating and rapidly recovering I(Ca,L) in the adult sinus node may contribute to the enhanced impulse initiation at this age in the absence of I(Na).     

Electrical remodeling in a canine model of ischemic cardiomyopathy

The nature of electrical remodeling in a canine model of ischemic cardiomyopathy (ICM; induced by repetitive intracoronary microembolizations) that exhibits spontaneous ventricular tachycardia is not entirely clear. We used the patch-clamp technique to record action potentials and ionic currents of left ventricular myocytes isolated from the region affected by microembolizations. We also used the immunoblot technique to examine channel subunit expression in adjacent affected tissue. Ventricular myocytes and tissue isolated from the corresponding region of normal hearts served as control. ICM myocytes had prolonged action potential duration (APD) and more pronounced APD dispersion. Slow delayed rectifier current (I(Ks)) was reduced at voltages positive to 0 mV, along with a negative shift in its voltage dependence of activation. Immunoblots showed that there was no change in KCNQ1.1 (I(Ks) pore-forming or alpha-subunit), but KCNE1 (I(Ks) auxiliary or beta-subunit) was reduced, and KCNQ1.2 (a truncated KCNQ1 splice variant with a dominant-negative effect on I(Ks)) was increased. Transient outward current (I(to)) was reduced, along with an acceleration of the slow phase of recovery from inactivation. Immunoblots showed that there was no change in Kv4.3 (alpha-subunit of fast-recovering I(to) component), but KChIP2 (beta-subunit of fast-recovering component) and Kv1.4 (alpha-subunit of slow-recovering component) were reduced. Inward rectifier current was reduced. L-type Ca current was unaltered. The immunoblot data provide mechanistic insights into the observed changes in current amplitude and gating kinetics of I(Ks) and I(to). We suggest that these changes, along with the decrease in inward rectifier current, contribute to APD prolongation in ICM hearts.     

AKAP proteins anchor cAMP-dependent protein kinase to KvLQT1/IsK channel complex

In cardiac myocytes, the slow component of the delayed rectifier K(+) current (I(Ks)) is regulated by cAMP. Elevated cAMP increases I(Ks) amplitude, slows its deactivation kinetics, and shifts its activation curve. At the molecular level, I(Ks) channels are composed of KvLQT1/IsK complexes. In a variety of mammalian heterologous expression systems maintained at physiological temperature, we explored cAMP regulation of recombinant KvLQT1/IsK complexes. In these systems, KvLQT1/IsK complexes were totally insensitive to cAMP regulation. cAMP regulation was not restored by coexpression with the dominant negative isoform of KvLQT1 or with the cystic fibrosis transmembrane regulator. In contrast, coexpression of the neuronal A kinase anchoring protein (AKAP)79, a fragment of a cardiac AKAP (mAKAP), or cardiac AKAP15/18 restored cAMP regulation of KvLQT1/IsK complexes inasmuch as cAMP stimulation increased the I(Ks) amplitude, increased its deactivation time constant, and negatively shifted its activation curve. However, in cells expressing an AKAP, the effects of cAMP stimulation on the I(Ks) amplitude remained modest compared with those previously reported in cardiac myocytes. The effects of cAMP stimulation were fully prevented by including the Ht31 peptide (a global disruptor of protein kinase A anchoring) in the intracellular medium. We concluded that cAMP regulation of I(Ks) requires protein kinase A anchoring by AKAPs, which therefore participate with the channel protein complex underlying I(Ks).     

Diverse phenotypes of outward currents in cells that have survived in the 5-day-infarcted heart

We have shown reduced density and altered kinetics in slowly activating K+ currents (I(Ks)) in epicardial border zone (EBZ) cells (IZs) of the 5-day-infarcted canine heart (Jiang M, Cabo, C, Yao J-A, Boyden PA, and Tseng G-N. Cardiovasc Res 48: 34-43, 2000). beta-Adrenergic stimulation with isoproterenol increases I(Ks) in normal cells (NZs). In this study, we used a voltage-clamp protocol with an external solution to isolate I(Ks) from contaminating currents to determine the effects of 1 muM isoproterenol on I(Ks) in IZs and NZs. Under our recording conditions, 10 microM azimilide-sensitive currents were stimulated with isoproterenol to compare responsiveness of I(Ks) to isoproterenol in the two cell groups. I(Ks) tail density was reduced 67% in IZs (group I, n = 26) compared with NZs (n = 24, P < 0.05). Isoproterenol-stimulated azimilide-sensitive tail currents were increased 1.72 +/- 0.2-fold in NZs and 2.2 +/- 0.3-fold in IZs (P > 0.05). In 33% of IZs (group II, n = 13), native currents showed no tail currents; however, isoproterenol-stimulated azimilide-sensitive currents were voltage dependent, fast activating, and large in amplitude compared with group I IZs, similar to "lone" KCNQ1 currents. Using short clamp pulses, we also found an increase in sustained currents sensitive to tetraethylammonium chloride (TEA) and no change in C-9356-sensitive currents in IZs with little or no transient outward current. In some IZs where I(Ks) is downregulated, the effect of isoproterenol on I(Ks) was similar to that on I(Ks) in NZs. In others, the existence of lone KCNQ1-type currents, which are sensitive to beta-adrenergic stimulation, is consistent with our findings of an increased KCNQ1-to-KCNE1 mRNA ratio (Jiang et al.). Accompanying altered I(Ks) in IZs are an enhanced TEA-sensitive current and a normal C-9356-sensitive current.     

Increasing I(Ks) corrects abnormal repolarization in rabbit models of acquired LQT2 and ventricular hypertrophy

Excessive action potential (AP) prolongation and early afterdepolarizations (EAD) are triggers of malignant ventricular arrhythmias. A slowly activating delayed rectifier K+ current (I(Ks)) is important for repolarization of ventricular AP. We examined the effects of I(Ks) activation by a new benzodiazepine (L3) on the AP of control, dofetilide-treated, and hypertrophied rabbit ventricular myocytes. In both control and hypertrophied myocytes, L3 activated I(Ks) via a negative shift in the voltage dependence of activation and a slowing of deactivation. L3 had no effect on L-type Ca(2+) current or other cardiac K+ currents tested. L3 shortened AP of control, dofetilide-treated, and hypertrophied myocytes more at 0.5 than 2 Hz. Selective activation of I(Ks) by L3 attenuates prolonged AP and eliminated EAD induced by rapidly activating delayed rectifier K+ current inhibition in control myocytes at 0.5 Hz and spontaneous EAD in hypertrophied myocytes at 0.2 Hz. Pharmacological activation of I(Ks) is a promising new strategy to suppress arrhythmias resulting from excessive AP prolongation in patients with certain forms of long QT syndrome or cardiac hypertrophy and failure.     

Contribution of L-type Ca2+ channels to early afterdepolarizations induced by I Kr and I Ks channel suppression in guinea pig ventricular myocytes

Early afterdepolarizations (EADs) induced by suppression of cardiac delayed rectifier I (Kr) and/or I (Ks) channels cause fatal ventricular tachyarrhythmias. In guinea pig ventricular myocytes, partial block of one of the channels with complete block of the other reproducibly induced EADs. Complete block of both I (Kr) and I (Ks) channels depolarized the take-off potential and reduced the amplitude of EADs, which in some cases were not clearly separated from the preceding action potentials. A selective L-type Ca(2+) (I (Ca,L)) channel blocker, nifedipine, effectively suppressed EADs at submicromolar concentrations. As examined with the action potential-clamp method, I (Ca,L) channels mediated inward currents with a spike and dome shape during action potentials. I (Ca,L) currents decayed mainly due to inactivation in phase 2 and deactivation in phase 3 repolarization. When EADs were induced by complete block of I (Kr) channels with partial block of I (Ks) channels, repolarization of the action potential prior to EAD take-off failed to increase I (K1) currents and thus failed to completely deactivate I (Ca,L) channels, which reactivated and mediated inward currents during EADs. When both I (Kr) and I (Ks) channels were completely blocked, I (Ca,L) channels were not deactivated and mediated sustained inward currents until the end of EADs. Under this condition, the recovery and reactivation of I (Ca,L) channels were absent before EADs. Therefore, an essential mechanism underlying EADs caused by suppression of the delayed rectifiers is the failure to completely deactivate I (Ca,L) channels.     

Specific serine proteases selectively damage KCNH2 (hERG1) potassium channels and I(Kr)

KCNH2 (hERG1) encodes the alpha-subunit proteins for the rapidly activating delayed rectifier K+ current (I(Kr)), a major K+ current for cardiac myocyte repolarization. In isolated myocytes I(Kr) frequently is small in amplitude or absent, yet KCNH2 channels and I(Kr) are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and I(Kr) are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myocytes I(Kr), slowly activating delayed rectifier K+ current (I(Ks)), and inward rectifier K+ current (I(K1)), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current (I(KCNH2)) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished I(Kr); and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of I(KCNH2) and its mature channel protein over several hours. Thus the channel protein for I(KCNH2) and I(Kr) is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low I(Kr) density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells.     

Tetrahydronaphthalene-derived amino alcohols and amino ketones as potent and selective inhibitors of the delayed rectifier potassium current IKs

Class III anti-arrhythmic drugs (e.g., dofetilide) prolong cardiac action potential duration (APD) by blocking the fast component of the delayed rectifier potassium current (I(Kr)). The block of I(Kr) can result in life threatening ventricular arrhythmias (i.e., torsades de pointes). Unlike I(Kr), the role of the slow component of the delayed rectifier potassium current (I(Ks)) becomes significant only at faster heart rate. Therefore selective blockers of I(Ks) could prolong APD with a reduced propensity to cause pro-arrhythmic side effects. This report describes structure-activity relationships (SARs) of a series of I(Ks) inhibitors derived from 6-alkoxytetralones with good in vitro activity (IC(50) > or =30 nM) and up to 40-fold I(Ks)/I(Kr) selectivity.     

A rapidly activating delayed rectifier K+ current regulates pacemaker activity in adult mouse sinoatrial node cells

We have investigated the physiological role of the "rapidly activating" delayed rectifier K+ current (IKr) in pacemaker activity in isolated sinoatrial node (SAN) myocytes and the expression of mouse ether-a-go-go (mERG) genes in the adult mouse SAN. In isolated, voltage-clamped SAN cells, outward currents evoked by depolarizing steps (greater than -40 mV) were strongly inhibited by the class III methanesulfonanilide compound E-4031 (1-2.5 microM), and the deactivation "tail" currents that occurred during repolarization to a membrane potential of -45 mV were completely blocked. E-4031-sensitive currents (IKr) reached a maximum at a membrane potential of -10 mV and showed pronounced inward rectification at more-positive membrane potentials. Activation of IKr occurred at -40 to 0 mV, with half-activation at about -24 mV. The contribution of IKr to action potential repolarization and diastolic depolarization was estimated by determining the E-4031-sensitive current evoked during voltage clamp with a simulated mouse SAN action potential. IKr reached its peak value (approximately 0.6 pA/pF) near -25 mV, close to the midpoint of the repolarization phase of the simulated action potential, and deactivated almost completely during the diastolic interval. E-4031 (1 microM) slowed the spontaneous pacing rate of Langendorff-perfused, isolated adult mouse hearts by an average of 36.5% (n = 5). Expression of mRNA corresponding to three isoforms coded by the mouse ERG1 gene (mERG1), mERG1a, mERG1a', and mERG1b, was consistently found in the SAN. Our data provide the first detailed characterization of IKr in adult mouse SAN cells, demonstrate that this current plays an important role in pacemaker activity, and indicate that multiple isoforms of mERG1 can contribute to native SAN IKr.     

Quantitative comparison of cardiac ventricular myocyte electrophysiology and response to drugs in human and nonhuman species

Explanations for arrhythmia mechanisms at the cellular level are usually based on experiments in nonhuman myocytes. However, subtle electrophysiological differences between species may lead to different rhythmic or arrhythmic cellular behaviors and drug response given the nonlinear and highly interactive cellular system. Using detailed and quantitatively accurate mathematical models for human, dog, and guinea pig ventricular action potentials (APs), we simulated and compared cell electrophysiology mechanisms and response to drugs. Under basal conditions (absence of β-adrenergic stimulation), Na(+)/K(+)-ATPase changes secondary to Na(+) accumulation determined AP rate dependence for human and dog but not for guinea pig where slow delayed rectifier current (I(Ks)) was the major rate-dependent current. AP prolongation with reduction of rapid delayed rectifier current (I(Kr)) and I(Ks) (due to mutations or drugs) showed strong species dependence in simulations, as in experiments. For humans, AP prolongation was 80% following I(Kr) block. It was 30% for dog and 20% for guinea pig. Under basal conditions, I(Ks) block was of no consequence for human and dog, but for guinea pig, AP prolongation after I(Ks) block was severe. However, with β-adrenergic stimulation, I(Ks) played an important role in all species, particularly in AP shortening at fast rate. Quantitative comparison of AP repolarization, rate-dependence mechanisms, and drug response in human, dog, and guinea pig revealed major species differences (e.g., susceptibility to arrhythmogenic early afterdepolarizations). Extrapolation from animal to human electrophysiology and drug response requires great caution.     

Gating of I(sK) channels expressed in Xenopus oocytes.

The channel underlying the slow component of the voltage-dependent delayed outward rectifier K+ current, I(Ks), in heart is composed of the minK and KvLQT1 proteins. Expression of the minK protein in Xenopus oocytes results in I(Ks)-like currents, I(sK), due to coassembly with the endogenous XKvLQT1. The kinetics and voltage-dependent characteristics of I(sK) suggest a distinct mechanism for voltage-dependent gating. Currents recorded at 40 mV from holding potentials between -60 and -120 mV showed an unusual "cross-over," with the currents obtained from more depolarized holding potentials activating more slowly and deviating from the Cole-Moore prediction. Analysis of the current traces revealed two components with fast and slow kinetics that were not affected by the holding potential. Rather, the relative contribution of the fast component decreased with depolarized holding potentials. Deactivation and reactivation, after a short period of repolarization (100 ms), was markedly faster than the fast component of activation. These gating properties suggest a physiological mechanism by which cardiac I(Ks) may suppress premature action potentials.