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 hKv1.5 channels by bupivacaine is stereoselective, with (R)-(+)-bupivacaine being 7-fold more potent than (S)-(-)-bupivacaine. The study of the effects of chemically related enantiomers on these channels may help to elucidate the structural determinants of stereoselective hKv1.5 channels block by local anesthetics. In this study, we analyzed the effects of (R)-(+)-ropivacaine, (R)-(+)-mepivacaine, and (S)-(-)-mepivacaine on hKv1.5 channels stably expressed in Ltk- cells. (R)-(+)-Ropivacaine inhibited hKv1.5 current and induced a fast initial decline superimposed to the slow inactivation during the application of depolarizing pulses, which reached steady state at the end of 250-msec depolarizing pulses. The concentration-dependence block induced by (R)-(+)-ropivacaine yielded a KD value of 32 +/- 1 microM [i.e., 2.5-fold more potent than (S)-(-)-ropivacaine]. (R)-(+)-Ropivacaine block also was voltage dependent, with a fractional electrical distance (delta) of 0.156 +/- 0.003 (n = 14) referred to the inner surface. Both (S)-(-)- and (R)-(+)-mepivacaine blocked hKv1.5 channels, with KD values of 286.8 +/- 34.1 and 379.0 +/- 56.0 microM, respectively [i.e., block was not stereoselective (p > 0.05)]. (S)-(-)-Mepivacaine and (R)-(+)-mepivacaine block displayed no apparent time-dependence due to a very fast dissociation rate constant. However, block by mepivacaine enantiomers was voltage dependent, with delta values of 0.154 +/- 0.015 and 0.160 +/- 0.008 for the (S)-(-)- and (R)-(+)-enantiomers, respectively. We conclude that (1) (R)-(+)-ropivacaine and mepivacaine enantiomers block the open state of hKv1.5 channels and (2) the length of their alkyl substituent at position 1 determines the potency and the degree of stereoselectivity.

These results demonstrated that at therapeutic concentrations, both losartan and E3174 modified the cardiac delayed rectifier hKv1.5, HERG, and Ks currents.

1 The goal of this study was to analyse the e ects of propafenone and its major metabolite, 5-hydroxypropafenone, on a human cardiac K + channel (hKv1.5) stably expressed in Ltk 7 cells and using the whole-cell con®guration 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 mM and 9.2+1.6 mM, 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)610 6 M 71 s 71 and 39.5+4.2 s 71 , respectively. For 5-hydroxy-propafenone these values averaged (2.3+0.3)610 6 M 71 s 71 and 21.4+3.1 s 71 , respectively. 5 Both drugs reduced the tail current amplitude recorded at 740 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 signi®cant 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 e ects are in the therapeutical range.

In the present study, we analyzed the effects of two angiotensin II type 1 receptor antagonists, candesartan (0.1 microM) and eprosartan (1 microM), on hKv1.5, HERG, KvLQT1+minK, and Kv4.3 channels expressed on Ltk(-) or Chinese hamster ovary cells using the patch-clamp technique. Candesartan and eprosartan produced a voltage-dependent block of hKv1.5 channels decreasing the current at +60 mV by 20.9 +/- 2.3% and 14.3 +/- 1.5%, respectively. The blockade was frequency-dependent, suggesting an open-channel interaction. Eprosartan inhibited the tail amplitude of HERG currents elicited on repolarization after pulses to +60 mV from 239 +/- 78 to 179 +/- 72 pA. Candesartan shifted the activation curve of HERG channels in the hyperpolarizing direction, thus increasing the current amplitude elicited by depolarizations to potentials between -50 and 0 mV. Candesartan reduced the KvLQT1+minK currents elicited by 2-s pulses to +60 mV (38.7 +/- 6.3%). In contrast, eprosartan transiently increased (8.8 +/- 2.7%) and thereafter reduced the KvLQT1+minK current amplitude by 17.7 +/- 3.0%. Eprosartan, but not candesartan, blocked Kv4.3 channels in a voltage-dependent manner (22.2 +/- 3.5% at +50 mV) without modifying the voltage-dependence of Kv4.3 channel inactivation. Candesartan slightly prolonged the action potential duration recorded in guinea pig papillary muscles at all driving rates. Eprosartan prolonged the action potential duration in muscles driven at 0.1 to 1 Hz, but it shortened this parameter at faster rates (2--3 Hz). All these results demonstrated that candesartan and eprosartan exert direct effects on Kv1.5, HERG, KvLQT1+minK, and Kv4.3 currents involved in human cardiac repolarization.