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

16 páginas, 8 figuras.[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.This study was supported by FIS grant 95/0318 (Dr Valenzuela), Salud 2000 grant (Dr Valenzuela), CICYT grant SAF92-0157 (Dr Tamargo), CAM grant 157/92 (Dr Tamargo), and NIH grant HL-47599 (Dr Snyders).Peer reviewe

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 μM and 3 μM, 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 μM) 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.This work was supported by FIS 95/0318 to C.V.., CICYT SAF96-0042 (to J.T.) and CAM 157/92 to (J.T.) Grants.Peer Reviewe

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); P0.05]. 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∼31 μM). [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.This work has been supported by HL47599 (D.J.S.), HL49330 (M.M.T.), HL46681 (D.J.S. and M.M.T.) from the National Institutes of Health, and by FIS 95/0318 (C.V.), CICYT SAF96-0042 (J.T.) and CICYT SAF98-0058 (C.V.) Grants.Peer Reviewe

Molecular determinants of stereoselective bupivacaine block of hKv1.5 channels

18 pages, 7 figures, 3 tables.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.This study was supported by FIS 95/0318 (Dr Valenzuela), CICYT SAF96–0042 (Dr Tamargo), and National Institute of Health grants HL-47599 (Dr Snyders), HL-46681 (Drs Snyders and Tamkun), and HL-49330 (Dr Tamkun).Peer reviewe

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 μM and 9.2±1.6 μM, 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)×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)×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