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.
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 wildtype 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.The slow delayed rectifier K ϩ current, I Ks , 1 is one of several outward currents that modulate repolarization of cardiac action potentials (1, 2). The channel mediating I Ks is formed by coassembly of two subunits, minK and KvLQT1 (3, 4). Mutations in KVLQT1 (also called KCNQ1) cause long QT syndrome (LQT) (5), an inherited disorder of ventricular repolarization that predisposes affected individuals to cardiac arrhythmias and sudden death (6). The physiologic consequences of some mutations have been studied by heterologous expression of KvLQT1 in Xenopus oocytes or mammalian cells (7-10). Most missense mutations in KVLQT1 cause loss of channel function when mutant subunits are expressed alone and a dominantnegative effect when coexpressed with wild-type (WT) KvLQT1 subunits. However, some mutations alter the function of Kv-LQT1 channels, and a study of their biophysical properties has led to novel insights into the structural basis of channel function. For example, the LQT-associated mutation V254M caused loss of function but accelerated activation of KvLQT1 channel current when mutant subunits were coexpressed with WT Kv-LQT1 subunits (8). V254M is located in the cytosolic loop connecting the S4 and S5 transmembrane domains of KvLQT1. Missense mutations in the S4-S5 linker have also been reported to accelerate activation and deactivation of human ether-a-go-go-related gene (HERG) channels (11). Furthermore, the S4-S5 linker determines the rate of deactivation of chimeric channels constructed from Kv2.1 and Kv3.1 (12). Together, these studies indicate th...
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.
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.
Background Zatebradine is a bradycardic agent that inhibits the hyperpolarization-activated current (I f ) in the rabbit
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