Aims A major problem in contemporary therapeutics is to predict those non-antiarrhythmic drugs (nards) which might prolong the QT interval. Block of repolarizing cardiac K+ current is the most likely cause of drug-induced QT prolongation. In this paper we compared six members from four important classes of nards, antihistamines, antipsychotics, antibiotics and prokinetics, for their block of the major repolarizing cardiac potassium currents I-Kr, I-Ks, I-To. and I-Kur. The currents were produced by heterologous expression of HERG (KCNH2), MinK/KvLQT1 (KCNE1/ KCNQ1), Kv4.3 (KCND3) and Kv1.5 (KCNA5) respectively. To evaluate the effects of different cellular backgrounds HERG was expressed stably in HEK 293 and mouse L cells, and transiently in Xenopus laevis oocytes.Methods and Results We measured currents with whole cell patch clamp and calculated IC50 values from dose-response curves at room and body temperatures. In all six cases HERG was the most sensitive target among the cloned K+ channels. HERG channels expressed in different mammalian cell lines had similar IC50 values. IC50 values were five to one hundred times larger when HERG was expressed transiently in Xenopus oocytes. Block was temperature-dependent but the effects were small and variable. For the nards terfenadine, sertindole and cisapride that have been withdrawn from the drug market, the IC50 values for HERG block were nanomolar, within the range for block of the primary target, and therefore within the therapeutic range.Conclusion Cloned ion channel assays are robust preclinical predictors of non-cardiac proarrhythmic drugs. (Eur Heart J Supplements 2001; 3 (Suppl K): K23-K30 (C) 2001 The European Society of Cardiology.

The rapid, repolarizing K(+) current in cardiomyocytes (I(Kr)) has unique inwardly rectifying properties that contribute importantly to the downstroke of the cardiac action potential. The human ether-à-go-go-related gene (HERG) expresses a macroscopic current virtually identical to I(Kr), but a description of the single-channel properties that cause rectification is lacking. For this reason we measured single-channel and macropatch currents heterologously expressed by HERG in Xenopus oocytes. Our experiments had two main findings. First, the single-channel current-voltage relation showed inward rectification, and conductance was 9.7 pS at -100 mV and 3.9 pS at 100 mV when measured in symmetrical 100 mM K(+) solutions. Second, single channels frequently showed no openings during depolarization but nevertheless revealed bursts of openings during repolarization. This type of gating may explain the inward rectification of HERG currents. To test this hypothesis, we used a three-closed state kinetics model and obtained rate constants from fits to macropatch data. Results from the model are consistent with rapid inactivation from closed states as a significant source of HERG rectification.

Low voltage-activated Ca2+ channels play important roles in pacing neuronal firing and producing network oscillations, such as those that occur during sleep and epilepsy. Here we describe the cloning and expression of the third member of the T-type family, alpha1I or CavT.3, from rat brain. Northern analysis indicated that it is predominantly expressed in brain. Expression of the cloned channel in either Xenopus oocytes or stably transfected human embryonic kidney-293 cells revealed novel gating properties. We compared these electrophysiological properties to those of the cloned T-type channels alpha1G and alpha1H and to the high voltage-activated channels formed by alpha1Ebeta3. The alpha1I channels opened after small depolarizations of the membrane similar to alpha1G and alpha1H but at more depolarized potentials. The kinetics of activation and inactivation were dramatically slower, which allows the channel to act as a Ca2+ injector. In oocytes, the kinetics were even slower, suggesting that components of the expression system modulate its gating properties. Steady-state inactivation occurred at higher potentials than any of the other T channels, endowing the channel with a substantial window current. The alpha1I channel could still be classified as T-type by virtue of its criss-crossing kinetics, its slow deactivation (tail current), and its small (11 pS) conductance in 110 mM Ba2+ solutions. Based on its brain distribution and novel gating properties, we suggest that alpha1I plays important roles in determining the electroresponsiveness of neurons, and hence, may be a novel drug target.

The use of nonsedating antihistamines may, on rare occasions, be associated with cardiac arrhythmias. This could be due to blockade of voltage-dependent K+ channels in the heart, leading to a prolongation in repolarization in the human myocardium. For this reason, we examined the effects of the nonsedating antihistamine loratadine on a rapidly activating delayed-rectifier K+ channel (Kv1.5) cloned from human heart and stably expressed in HEK 293 cells or mouse Ltk- cells. Using patch-clamp electrophysiology, we found that loratadine blocked Kv1.5 current measured from inside-out membrane patches at concentrations of > or = 100 nM, resulting in an IC50 value of 808 nM at +50 mV. The drug enhanced the rate of Kv1.5 current decay, and block was enhanced at membrane potentials near threshold relative to higher potentials. Loratadine did not alter the kinetics of Kv1.5 current activation or deactivation. Unitary Kv1.5 currents were recorded in cell-attached patches. At the single-channel level, the main effect of loratadine was to reduce the mean probability of opening of Kv1.5. This effect of loratadine was achieved by a reduced number of openings in bursts and burst duration. Finally, loratadine (10 microM) failed to inhibit HERG K+ channel currents expressed in Xenopus laevis oocytes. It is concluded that loratadine is an effective blocker of Kv1.5 that interacts with an activated state or states of the channel. This interaction suggests a potential for loratadine to alter cardiac excitability in vivo.

Dofetilide, a methanesulfonanilide derivative, is a potent class III antiarrhythmic drug. Like other members of this class of K+ channel blockers, the sites in the channel to which the drug binds are unknown, although high and low affinity binding has been reported in cardiomyocytes. The most sensitive K+ channel target for dofetilide seems to be IKr, the rapid component of the repolarizing delayed rectifier K+ current. However, block of other K+ channels occurs at higher concentrations and is of special interest in regard to toxicity. Recently, we have demonstrated that hIRK, a cloned inward rectifier K+ channel (IRK) isolated from human atrium and expressed heterologously in Xenopus oocytes, is blocked by dofetilide. We report the localization of a site that is critical for dofetilide block in hIRK. We used chimeric constructs between hIRK and ROMK1, a related inward rectifier that is drug resistant. Substitution of hIRK-M2, the second putative transmembrane spanning segment of IRKs, with ROMK1-M2 increased unblocking of dofetilide by 10-20-fold in hIRK. Site-directed mutagenesis further pinpointed the effects to a single hydrophobic residue (I177) in M2. A reduction in hydrophobicity by the point mutation I177C increased recovery from block > 10-fold (1.17 sec in wild-type to 0.112 sec at -80 mV at physiological K+ concentrations), leading us to suggest that hydrophobic interactions are essential for dofetilide block in hIRK. A similar mechanism may explain dofetilide block in other ion channels, including IKr.

BACKGROUND:The human ether-a-go-go-related gene (HERG) is one locus for the hereditary long-QT syndrome. A hypothesis is that HERG produces the repolarizing cardiac potassium current IKr with the consequence that mutations in HERG prolong the QT interval by reducing IKr. The elementary properties of HERG are unknown, and as a test of the hypothesis that HERG produces IKr, we compared their elementary properties.METHODS AND RESULTS:We injected HERG cRNA into Xenopus oocytes and measured currents from single channels or current variance from the noise produced by ensembles of channels recorded from macro patches. Single-channel conductance was dependent on the extracellular potassium concentration ([K]o). At physiological [K]o, it was 2 picosiemens (pS), and at 100 mmol/L [K]o, it was 10 pS. Openings occurred in bursts with a mean duration of 26 ms at -100 mV. Mean open time was 3.2 ms and closed times were 1.0 and 26 ms. In excised macro patches, HERG currents were blocked by the class III antiarrhythmic drug dofetilide, with an IC50 of 35 nmol/L. Dofetilide block was slow and greatly attenuated at positive potentials at which HERG rectifies.CONCLUSIONS:The microscopic physiology of HERG and IKr is similar, consistent with HERG being an important component of IKr. The pharmacology is also similar; dofetilide appears to primarily block activated channels and has a much lower affinity for closed and inactivated channels.

The loci for inactivation in calcium channel proteins are unknown. Mechanisms for inactivation may be distributed across Ca 2+ channel subunits and appear to be complex, multiple and interacting. We took advantage of the properties of chimeras, constructed between cardiac (H4) and skeletal muscle (Sk4) calcium channel α 1 subunits to study the molecular mechanism of inactivation in L‐type calcium channels. Sk1H3, a chimeric construct of these two L‐type calcium channels, was expressed in Xenopus oocytes in the absence of auxiliary subunits. Sk1H3 incorporated repeat I from skeletal muscle α 1 , and repeats II, III, IV from heart α 1 , subunit. Sk1H3 inactivated faster ( τ ≈ 300 ms) and more fully than the wild‐type H4 with Ba 2+ ions as the charge carrier. Thus, inactivation of Sk1H3 was 90% complete after a 5‐s conditioning pulse at +20 mV while inactivation of H4 was only 37% complete. Sk1H3 inactivation also developed at more negative potentials with E 0.5 = −15 mV as compared to E 0.5 = −5 mV for H4. In the presence of external calcium ions, the extent of inactivation significantly increased from 37 to 83% for H4 while inactivation of Sk1H3 was only slightly increased. Inactivation with Ba 2+ as the charge carrier was confirmed at the single‐ channel level where averaged single‐channel ensembles showed a similar rate of inactivation. Collectively, these observations demonstrate that Sk1H3 inactivation appears to have a prominent voltage‐dependent component. Whether Sk1H3 inactivation involves interactions within repeat I alone or interactions between repeat I and site(s) located in the three other repeats of the α 1 subunit has yet to be determined.

Voltage-sensitive Ca2+ channels are multisubunit complexes that include, among others, a large alpha 1 subunit, which by itself is sufficient to form a channel. Several alpha 1 genes encoding L-, N-, and P-type Ca2+ channels have been cloned. These alpha 1 genes share a high degree of sequence homology in the putative transmembrane regions, but vary substantially in the putative intracellular loops and the flanking amino and carboxyl termini. In the present study, we investigated the functional roles of the 665-amino acid long carboxyl terminus of a cardiac alpha 1 by constructing deletion mutants. Expression in Xenopus oocytes of delta C1856, delta C1733, and delta C1700, which lack from 307 to 472 amino acids at the carboxyl terminus, led to inward Ba2+ currents that were 4- to 6-fold greater than observed with the 2171-amino acid long wild type alpha 1. Ionic currents increased without a change in the amount of charge moved during voltage-dependent gating, suggesting that the increase in ionic currents was not due to an increase in the number of channels that were expressed. Single channel analysis revealed an unaltered unitary conductance. Thus, removal of up to 70% of the carboxyl terminus increased current density by facilitating the coupling between the voltage-dependent gating and channel opening, leading to an increased opening probability of the channel.

We used amplifying effects of calcium channel beta subunits to identify endogenous calcium channels in Xenopus oocytes. Expression of rat brain beta 4 increased macroscopic endogenous current magnitude with a small effect on kinetics. In contrast, expression of rat brain/cardiac beta 2 produced a much larger increase in current magnitude and dramatically slowed current decay. Low concentrations of omega-conotoxin GVIA irreversibly blocked currents in both uninjected and beta 2-injected oocytes. Single channel recordings revealed both T- and N-type calcium channels with conductances of 9 and 18 pS, respectively, in uninjected oocytes and in oocytes expressing either beta subunit. Expression of either beta subunit slowed average current decay of T-type single channels. Slowing of T-type current decay by expression of beta 2 was due to reopening of the channels. N-type single channel average current decay showed little change with expression of beta 4, whereas expression of beta 2 slowed average current decay.

Prolongation of the QT interval corrected for heart rate (QTc) can lead to the development of torsades de pointes, a life-threatening form of polymorphic ventricular tachycardia. However, the QTc interval duration exhibits a high degree of spontaneous variability and is not necessarily a direct predictor of the risk of torsades. This observation holds implications for the assessment of the potential proarrhythmic effects of noncardiac pharmacologic agents. To date, the antihistamine terfenadine is the only noncardiac drug that has undergone a comprehensive and systematic evaluation related to the consequences of its causing QTc prolongation. The results suggest that QTc prolongation resulting solely from terfenadine at clinical doses does not have an important impact on clinically relevant endpoints. The risk of serious ventricular arrhythmias with terfenadine using epidemiologic data is the same or less than that associated with traditional first-generation antihistamines. The risk of a clinical cardiac event (QTc prolongation, ventricular arrhythmias, syncope, or sudden death) with terfenadine is similar to that of other antihistamines. Factors associated with increased risk in patients taking terfenadine include significant liver disease, hypokalemia, overdose, and concomitant administration of ketoconazole-like agents or erythromycin; use of terfenadine is relatively contraindicated in these settings. No increased risk of serious arrhythmias has been confirmed in conjunction with the use of terfenadine in patients with cardiac disease.

High threshold L-type Ca2+ channels of skeletal muscle are thought to consist of a complex of alpha 1, alpha 2 delta, beta, and gamma subunits. Expression of the cloned alpha 1 subunit from skeletal and cardiac muscle has established that this protein is the dihydropyridine-sensitive ion-conducting subunit. However, the kinetics of the skeletal muscle alpha 1 alone expressed in mouse L-cells were abnormally slow and were accelerated to within the normal range by coexpression with the skeletal muscle beta subunit. The kinetics of cardiac muscle alpha 1 were also slowed but to a lesser extent and were not altered by coexpression with skeletal muscle alpha 2. We show here that coexpression of the skeletal muscle beta subunit with the cardiac alpha 1 subunit in Xenopus laevis oocytes produced: 1) an increase in the peak voltage-sensitive current, 2) a shift of the peak current-voltage relationship to more hyperpolarized potentials, and 3) an increase in the rate of activation. Coexpression of the skeletal muscle gamma subunit did not have a significant effect on currents elicited by alpha 1. However, when gamma was coexpressed with beta and alpha 1, both peak currents and rates of activation at more negative potentials were increased. These results indicate that rather than simply amplifying expression of alpha 1, heterologous skeletal muscle beta and gamma subunits can modulate the biophysical properties of cardiac alpha 1.

We have tested the effects of the active 1-34 amino acid sequence of rat parathyroid hormone (PTH) on Ca2+ channel activity in neonatal rat ventricular cells. Rat PTH (30 pM to 10 nM) increased depolarization-induced Ca2+ influx into these cells, an effect that was abolished by 1 microM nifedipine. The 1-34 amino acid sequence of bovine PTH also stimulated Ca2+ influx in control cells but not in cells pretreated with cholera toxin. Rat PTH also elevated adenosine 3',5'-cyclic monophosphate accumulation in these ventricular myocytes. Whole cell voltage-clamp recordings confirmed a stimulatory effect of rat PTH on cardiac L-type Ca2+ channels. Cell-attached single channel recordings revealed an increase in the probability of channel opening as the primary mechanism for the enhancement of Ca2+ current. Taken together these results suggest an important role for PTH as an endogenous modulator of cardiac L-type Ca2+ channels.