Long QT syndrome-associated mutations in the S4-S5 linker of KvLQT1 potassium channels modify gating and interaction with minK subunits.

Long QT syndrome is an inherited disorder of cardiac repolarization caused by mutations in cardiac ion channel genes, including KVLQT1. In this study, the functional consequences of three long QT-associated missense mutations in KvLQT1 (R243C, W248R, E261K) were characterized using the Xenopus oocyte heterologous expression system and two-microelectrode voltage clamp techniques. These mutations are located in or near the intracellular linker between the S4 and S5 transmembrane domains, a region implicated in activation gating of potassium channels. The E261K mutation caused loss of function and did not interact with wild-type KvLQT1 subunits. R243C or W248R KvLQT1 subunits formed functional channels, but compared with wild-type KvLQT1 current, the rate of activation was slower, and the voltage dependence of activation and inactivation was shifted to more positive potentials. Co expression of minK and KvLQT1 channel subunits induces a slow delayed rectifier K(+) current, I(Ks), characterized by slow activation and a markedly increased magnitude compared with current induced by KvLQT1 subunits alone. Coexpression of minK with R243C or W248R KvLQT1 subunits suppressed current, suggesting that coassembly of mutant subunits with minK prevented normal channel gating. The decrease in I(Ks) caused by loss of function or altered gating properties explains the prolonged QT interval and increased risk of arrhythmia and sudden death associated with these mutations in KVLQT1.

Functional expression of an inactivating potassium channel (Kv4.3) in a mammalian cell line.

OBJECTIVE: The goal of this study was to characterize the electrophysiological properties of the Kv4.3 channels expressed in a mammalian cell line. METHODS: Currents were recorded using the whole-cell voltage clamp technique. RESULTS: The threshold for activation of the expressed Kv4.3 current was approximately -30 mV. The dominant time constant for activation was 1.71 +/- 0.16 ms (n = 10) at +60 mV. The current inactivated, this process being incomplete, resulting in a sustained level which contributed 15 +/- 2% (n = 25) of the total current. The time course of inactivation was fit by a biexponential function, the fast component contributing 74 +/- 5% (n = 9) to the overall inactivation. The fast time constant was voltage-dependent [27.6 +/- 2.0 ms at +60 mV (n = 10) versus 64.0 +/- 3.6 ms at 0 mV (n = 10); P < 0.01], whereas the slow was voltage-independent [142 +/- 15 ms at +60 mV (n = 10) versus 129 +/- 33 ms at 0 mV (n = 6) P > 0.05]. The voltage-dependence of inactivation exhibited midpoint and slope values of -26.9 +/- 1.5 mV and 5.9 +/- 0.3 mV (n = 21). Recovery from inactivation was faster at more negative membrane potentials [203 +/- 17 ms (n = 13) and 170 +/- 19 ms (n = 4), at -90 and -100 mV]. Bupivacaine block of Kv4.3 channels was not stereoselective (KD approximately 31 microM). CONCLUSIONS: The functional profile of Kv4.3 channels expressed in Ltk- cells corresponds closely to rat ITO, although differences in recovery do not rule out association with accessory subunits. Nevertheless, the sustained component needs to be considered with respect to native ITO.

Effects of propafenone and 5-hydroxy-propafenone on hKv1.5 channels.

1. The goal of this study was to analyse the effects of propafenone and its major metabolite, 5-hydroxy-propafenone, on a human cardiac K+ channel (hKv1.5) stably expressed in Ltk- cells and using the whole-cell configuration of the patch-clamp technique. 2. Propafenone and 5-hydroxy-propafenone inhibited in a concentration-dependent manner the hKv1.5 current with K(D) values of 4.4+/-0.3 microM and 9.2+/-1.6 microM, respectively. 3. Block induced by both drugs was voltage-dependent consistent with a value of electrical distance (referenced to the cytoplasmic side) of 0.17+/-0.55 (n=10) and 0.16+/-0.81 (n=16). 4. The apparent association (k) and dissociation (l) rate constants for propafenone were (8.9+/-0.9) x 10(6) M(-1) s(-1) and 39.5+/-4.2 s(-1), respectively. For 5-hydroxy-propafenone these values averaged (2.3+/-0.3) x 10(6) M(-1) s(-1) and 21.4+/-3.1 s(-1), respectively. 5. Both drugs reduced the tail current amplitude recorded at -40 mV after 250 ms depolarizing pulses to +60 mV, and slowed the deactivation time course resulting in a 'crossover' phenomenon when the tail currents recorded under control conditions and in the presence of each drug were superimposed. 6. Both compounds induced a small but statistically significant use-dependent block when trains of depolarizations at frequencies between 0.5 and 3 Hz were applied. 7. These results indicate that propafenone and its metabolite block hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner and the concentrations needed to observe these effects are in the therapeutical range.

Molecular determinants of stereoselective bupivacaine block of hKv1.5 channels.

Enantiomers of local anesthetics are useful probes of ion channel structure that can reveal three-dimensional relations for drug binding in the channel pore and may have important clinical consequences. Bupivacaine block of open hKv1.5 channels is stereoselective, with the R(+)-enantiomer being 7-fold more potent than the S(-)-enantiomer (Kd = 4.1 mumol/L versus 27.3 mumol/L). Using whole-cell voltage clamp of hKv1.5 channels and site-directed mutants stably expressed in Ltk- cells, we have identified a set of amino acids that determine the stereoselectivity of bupivacaine block. Replacement of threonine 505 by hydrophobic amino acids (isoleucine, valine, or alanine) abolished stereoselective block, whereas a serine substitution preserved it [Kd = 60 mumol/L and 7.4 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. A similar substitution at the internal tetraethylammonium binding site (T477S) reduced the affinity for both enantiomers similarly, thus preserving the stereoselectivity [Kd = 45.5 mumol/L and 7.8 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. Replacement of L508 or V512 by a methionine (L508M and V512M) abolished stereoselective block, whereas substitution of V512 by an alanine (V512A) preserved it. Block of Kv2.1 channels, which carry valine, leucine, and isoleucine residues at T505, L508, and V512 equivalent sites, respectively, was not stereoselective [Kd = 8.3 mumol/L and 13 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. These results suggest that (1) the bupivacaine binding site is located in the inner mouth of the pore, (2) stereoselective block displays subfamily selectivity, and (3) a polar interaction with T505 combined with hydrophobic interactions with L508 and V512 are required for stereoselective block.

Block of human cardiac Kv1.5 channels by loratadine: voltage-, time- and use-dependent block at concentrations above therapeutic levels.

OBJECTIVE: The aim of this study was to analyze the effects of loratadine on a human cardiac K+ channel (hKv1.5) cloned from human ventricle and stably expressed in a mouse cell line. METHODS: Currents were studied using the whole-cell configuration of the patch-clamp technique in Ltk- cells transfected with the gene encoding hKv1.5 channels. RESULTS: Loratadine inhibited in a concentration-dependent manner the hKv1.5 current, the apparent affinity being 1.2 +/- 0.2 microM. The blockade increased steeply between -30 and 0 mV which corresponded with the voltage range for channel opening, thus suggesting that the drug binds preferentially to the open state of the channel. The apparent association and dissociation rate constants were (3.6 +/- 0.5) x 10(6).M-1.s-1 and 3.7 +/- 1.6.s-1, respectively. Loratadine, 1 microM, increased the time constant of deactivation of tail currents elicited on return to -40 mV after 500 ms depolarizing pulses to +60 mV from 36.2 +/- 3.4 to 64.9 +/- 3.6 ms (n = 6, P < 0.01), thus inducing a 'crossover' phenomenon. Application of trains of pulses at 1 Hz lead to a progressive increase in the blockade reaching a final value of 48.6 +/- 4.3%. Recovery from loratadine-induced block at -80 mV exhibited a time constant of 743.0 +/- 78.0 ms. Finally, the results of a mathematical stimulation of the effects of loratadine, based on an open-channel block model, reproduced fairly well the main effects of the drug. CONCLUSIONS: The present results demonstrated that loratadine blocked hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner but only at concentrations much higher than therapeutic plasma levels in man.

Comparative effects of nonsedating histamine H1 receptor antagonists, ebastine and terfenadine, on human Kv1.5 channels.

The effects of ebastine and terfenadine, long-acting nonsedating histamine H1 receptor antagonists, were studied on hKv1.5 channels using the whole-cell voltage-clamp configuration of the patch-clamp technique in Ltk- cells transfected with the gene encoding the hKv1.5 channel. Upon depolarization to +60 mV, terfenadine, 1 microM and 3 microM, inhibited the hKv1.5 current by 42.4 +/- 6.4% and 69.3 +/- 4.2% (P < 0.01). In contrast, at the same range of concentrations, ebastine-induced inhibition of this K+ current averaged 6.5 +/- 2.0% and 13.0 +/- 2.0 (P < 0.05). At the highest concentration tested (3 microM) neither terfenadine carboxylate nor carebastine significantly modified hKv1.5 current. All these results suggest that ebastine could represent a safer alternative to terfenadine in the clinical practice.

Effects of ropivacaine on a potassium channel (hKv1.5) cloned from human ventricle.

BACKGROUND: Ropivacaine, a new amide local anesthetic agent chemically related to bupivacaine, is able to induce early after depolarizations in isolated cardiac preparations. The underlying mechanism by which ropivacaine induces this effect has not been explored, but it is likely to involve K+ channel block. METHODS: Cloned human cardiac K+ channels (hKv1.5) were stably transfected in Ltk cells, and the effects of ropivacaine on the expressed hKv1.5 currents were assessed using the whole-cell configuration of the patch-clamp technique. RESULTS: Ropivacaine (100 microM) did not modify the initial activation time course of the current, but induced a fast subsequent decline to a lower steady-state current level with a time constant of 12.2 +/- 0.6 ms. Ropivacaine inhibited hKv1.5 with an apparent KD of 80 +/- 4 microM. Block displayed an intrinsic voltage-dependent, consistent with an electrical distance for the binding site of 0.153 +/- 0.007 (n = 6) (from the cytoplasmic side). Ropivacaine reduced the tail current amplitude recorded at -40 mV, and slowed the deactivation time course, resulting in a "crossover" phenomenon when control and ropivacaine tail currents were superimposed. CONCLUSIONS: These results indicate that: (1) ropivacaine is an open channel blocker of hKv1.5; (2) binding occurs in the internal mouth of the ion pore; and (3) unbinding is required before the channel can close. These effects explain the ropivacaine availability of induction early after depolarizations and could be clinically relevant.

Effect of descarboethoxyloratadine, the major metabolite of loratadine, on the human cardiac potassium channel Kv1.5.

The effects of descarboethoxyloratadine (DCL), the major metabolite of loratadine, were studied on a human cardiac K+ channel (hKv1.5) cloned from human ventricle and stably expressed in a mouse cell line by means of the patch-clamp technique. DCL (1-100 microM) inhibited hKv1.5 current in a concentration-dependent manner with an apparent affinity constant of 12.5+/-1.2 microM. The blockade increased steeply over the voltage range of channel opening, which indicated that DCL binds preferentially to the open state of the channel. At more depolarized potentials a weaker voltage-dependence was observed consistent with a binding reaction sensing approximately 20% of the transmembrane electrical field. DCL, 20 microM, increased the time constant of deactivation of tail currents, thus inducing a 'crossover' phenomenon. The present results demonstrated that DCL blocked hKv1.5 channels in a concentration-, voltage-, and time-dependent manner.

Class III antiarrhythmic effects of zatebradine. Time-, state-, use-, and voltage-dependent block of hKv1.5 channels.

BACKGROUND: Zatebradine is a bradycardic agent that inhibits the hyperpolarization-activated current (I(f)) in the rabbit sinoatrial node. It also prolongs action potential duration in papillary muscles in guinea pigs and in Purkinje fibers in rabbits. The underlying mechanism by which zatebradine induces this effect has not been explored, but it is likely to involve K+ channel block. METHODS AND RESULTS: Cloned human cardiac K+ delayed rectifer currents (hKv1.5) were recorded in Ltk- cells transfected with their coding sequence. Zatebradine 10 mumol/L did not modify the initial activation time course of the current but induced a subsequent decline to a lower steady-state current level with a time constant of 109 +/- 16 ms. Zatebradine inhibited hKv1.5 with an apparent KD of 1.86 +/- 0.14 mumol/L. Block was voltage dependent (electrical distance delta = 0.177 +/- 0.003) and accumulated in a use-dependent manner during 0.5- and 1-Hz pulse trains because of slower recovery kinetics in the presence of the drug. Zatebradine reduced the tail current amplitude, recorded at -30 mV, and slowed the deactivation time course, which resulted in a "crossover" phenomenon when control and zatebradine tail currents were superimposed. CONCLUSIONS: These results indicate that (1) zatebradine is an open-channel blocker of hKv 1.5, (2) binding occurs in the internal mouth of the ion pore, (3) unbinding is required before the channel can close, and (4) zatebradine-induced block is use dependent because of slower recovery kinetics in the presence of the drug. These effects may explain the prolongation of the cardiac action potential and could be clinically relevant.

Mechanisms of block of a human cloned potassium channel by the enantiomers of a new bradycardic agent: S-16257-2 and S-16260-2.

1. The effects of S-16257-2 (S57) and S-16260-2 (R60), the two enantiomers of a new bradycardic agent, were studied on human cloned K+ channels (hKv1.5) stably expressed in a mouse L cell line using the whole-cell configuration of the patch-clamp technique. 2. S57 and R60 did not modify the sigmoidal activation time course of the current but reduced the amplitude and increased the rate of the decay of the current during the application of depolarizing pulses. Both, S57 and R60 produced a concentration-dependent block of hKv1.5 channels with apparent KD values of 29.0 +/- 1.9 microM and 40.9 +/- 4.0 microM, respectively. Thus, S57 was 1.4 fold more potent than R60 in blocking hKv1.5 channels. 3. The blockade produced by S57 and R60 was voltage-dependent and increased steeply between -30 and 0 mV, which corresponded with the voltage range for channel opening. This result indicated that both enantiomers block the hKv1.5 channels, preferentially, when they are in the open state. Between 0 and +60 mV the blockade exhibited a shallow voltage-dependence which was described by an electrical distance of 0.18 +/- 0.002 and 0.19 +/- 0.004 for S57 and R60, respectively. 4. S57 and R60 also increased the rate of decline of the current during the application of depolarizing pulses. The time constant of such decline (tau Block) was faster in the presence of R60 than in the presence of S57 (16.2 +/- 1.5 ms vs. 24.0 +/- 2.6 ms; P < 0.01). The apparent association rate constants (k) were similar for S57 and R60 ((0.52 +/- 0.13) x 10(6) M-1 s-1 and (0.66 +/- 0.13) x 10(6) M-1 s-1, respectively), whereas the dissociation rate constant (l) was faster for R60 than for S57 (25.8 +/- 1.8 s-1 and 13.0 +/- 2.4 s-1, respectively). 5. Both enantiomers slowed the deactivation of the tail currents elicited upon repolarization to -40 mV, thus inducing a 'crossover' phenomenon. These results suggested that drug unbinding is required before hKv1.5 channels can close. 6. It is concluded that R60 and S57 produced a similar time- voltage- and state-dependent block of hKv1.5 channels that can be interpreted as open channel block by the charged form of each enantiomer. The main difference between R60 and S57 were linked to the apparent dissociation rate constants.

Electromechanical effects of zatebradine on isolated guinea pig cardiac preparations.

The effects of zatebradine on rate and contractile force and transmembrane action potentials were studied in isolated guinea pig atria and ventricular papillary muscles. In spontaneously beating right atria, zatebradine, 10(-7)-10(-4) M, produced a negative chronotropic effect (IC50 = 6.5 +/- 3.0 x 10(-6) M) and prolonged the recovery of the sinus function. In addition, it produced a biphasic effect on the atrial contractility, so that at concentrations up to 10(-5) M, it exerted a positive inotropic effect, whereas at higher concentrations, a negative inotropic effect was observed (IC50 = 9.0 +/- 0.3 x 10(-5) M). In electrically driven left atria, zatebradine produced a negative inotropic effect, though no changes were observed in the total contraction time or the time to peak tension. In papillary muscles, zatebradine > or = 5 x 10(-6) M caused a significant decrease in the maximum upstroke velocity (Vmax) without altering the resting membrane potential and exerted biphasic effects on the action potential duration (APD). At concentrations up to 5 x 10(-5) M, it prolonged the APD, whereas at higher concentrations, it shortened the APD. In addition, zatebradine, 10(-4) M, significantly reduced the amplitude and Vmax of the slow action potentials elicited by isoproterenol in K(+)-depolarized papillary fibres. In the presence of zatebradine, trains of stimuli at rates between 0.5 and 3 Hz led to an exponential decline in Vmax (frequency-dependent Vmax block), which was augmented at higher rates of stimulation. The time constant for the recovery of Vmax from the frequency-dependent block was 2.9 s.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the two enantiomers, S-16257-2 and S-16260-2, of a new bradycardic agent on guinea-pig isolated cardiac preparations.

1. The electromechanical effects of two enantiomers, S-16257-2 (S57) and S-16260-2 (R60), were studied and compared in guinea-pig isolated atria and ventricular papillary muscles. The possible stereoselectivity of the interaction on the cardiac Na+ channel was analysed by comparing the effects of the two enantiomers on the onset and recovery kinetics of the frequency-dependent Vmax block. 2. In spontaneously beating right atria, S57 and R60 (10(-8)M-10(-4M) exerted a negative chronotropic effect (pIC50 = 5.07 +/- 0.19 and 4.76 +/- 0.18, respectively) and prolonged the sinus node recovery time, this effect being more marked with S57. In electrically driven left atria, S57 decreased (P < 0.05) contractile force only at 10(-4M) and R60 at concentrations > or = 5 x 10(-5M), whereas in papillary muscles the negative inotropic effect appeared at concentrations > 10(-5M). 3. In papillary muscles driven at 1 Hz, S57 and R60 at concentrations higher than 5 x 10(-6M) produced a concentration-dependent decrease in the maximum upstroke velocity (Vmax) and amplitude of the cardiac action potential without altering the resting membrane potential or the action potential duration. S57 and R60 had no effect on the characteristics of the slow action potentials elicited by isoprenaline in ventricular muscle fibres depolarized in high K+ (27 mM) solution. 4. At 5 x 10(-5M), S57 and R60 produced a small tonic Vmax block. However, in muscles driven at rates between 0.5 and 3 Hz both enantiomers produced an exponential decline in Vmax (frequency-dependent Vmax block) which augmented at higher rates of stimulation. The onset and offset rates of the frequency-dependent Vmax block were similar for both drugs. Both S57 and R60 prolonged the recovery time constant from the frequency-dependent block from 20.1 +/- 2.9 ms to 2-3 s.5. At 5 x 10-5 M, S57 and R60 shifted the membrane responsiveness curve in a hyperpolarizing direction.6. It can be concluded that S57 and R60, the two enantiomers of the new bradycardic agent, produced a similar frequency-dependent Vmax block which indicated that the interaction with the Na+ channel was not stereospecific. The analysis of the onset and offset kinetics of the frequency-dependent Vmax block suggested that both enantiomers can be considered as Na+ channel blockers with intermediate kinetics,e.g., class IA antiarrhythmic drugs.