Stereoselective block of a human cardiac potassium channel (Kv1.5) by bupivacaine enantiomers

Abstract

Stereoselective drug-channel interactions may help to elucidate the molecular basis of voltage-gated potassium channel block by local anesthetic drugs. We studied the effects of the enantiomers of bupivacaine on a cloned human cardiac potassium channel (hKv1.5). This channel was stably expressed in a mouse Ltk- cell line and studied using the whole-cell configuration of the patch-clamp technique. Both enantiomers modified the time course of this delayed rectifier current. Exposure to 20 microM of either S(-)-bupivacaine or R(+)-bupivacaine did not modify the activation time constant of the current, but reduced the peak outward current and induced a subsequent exponential decline of current with time constants of 18.7 +/- 1.1 and 10.0 +/- 0.9 ms, respectively. Steady-state levels of block (assessed with 250-ms depolarizing pulses to +60 mV) averaged 30.8 +/- 2.5% (n = 6) and 79.5 +/- 3.2% (n = 6) (p < 0.001), for S(-)- and R(+)-bupivacaine, respectively. The concentration dependence of hKv1.5 inhibition revealed apparent KD values of 27.3 +/- 2.8 and 4.1 +/- 0.7 microM for S(-)-bupivacaine and R(+)-bupivacaine, respectively, with Hill coefficients close to unity, suggesting that binding of one enantiomer molecule per channel was sufficient to block potassium permeation. Analysis of the rate constants of association (k) and dissociation (l) yielded similar values for l (24.9 s-1 vs. 23.6 s-1 for S(-)- and R(+)-bupivacaine, respectively) but different association rate constants (1.0 x 10(6) vs. 4.7 x 10(6) M-1 s-1 for S(-)- and R(+)-bupivacaine, respectively). Block induced by either enantiomer displayed a shallow voltage dependence in the voltage range positive to 0 mV, i.e., where the channel is fully open, consistent with an equivalent electrical distance delta of 0.16 +/- 0.01. This suggested that at the binding site, both enantiomers of bupivacaine experienced 16% of the applied transmembrane electrical field, referenced to the inner surface. Both bupivacaine enantiomers reduced the tail current amplitude recorded on return to -40 mV and slowed their time course relative to control, resulting in a "crossover" phenomenon. These data indicate 1) the charged form of both bupivacaine enantiomers block the hKv1.5 channel after it opens, 2) binding occurs within the transmembrane electrical field, 3) unbinding is required before the channel can close, 4) block of hKv1.5 channels by bupivacaine is markedly stereoselective, with the R(+)-enantiomer being the more potent one, 5) this stereoselective block was associated with a 1.11 -kcal/mol difference in binding energy between both enantiomers, and 6) the stereoselectivity derives mainly from a difference in the association rate constants, suggesting that the S(-)-enantiomer is less likely to access the binding site in an optimal configuration.

Effect of fluoxetine on a neuronal, voltage‐dependent potassium channel (Kv1.1)

“…As a consequence, the steepness of the voltage-dependence of open channel block will be highest at V test -values close to V 1/2 of the activation curve and lowest at V test values where all channels are activated. This phenomenon has clearly been observed for stereoselective block of hKv1.5 by bupivacaine enantiomers (Valenzuela et al, 1995), but is absent in our study on Flu (Figure 4b). One explanation could be that binding of Flu on RCK1 is entirely voltage-independent.…”

Mechanism underlying bupivacaine inhibition of G protein-gated inwardly rectifying K + channels

“…The levels of bupivacaine in blood (34-83 M) that produce seizures in sheep (45) are close to the K i determined for bupivacaine inhibition of GIRK channels (Ϸ20 M). Some types of voltagegated K ϩ channels are also inhibited by bupivacaine (4)(5)(6). Thus, the suppression of GIRK channel activity together with other K ϩ channels may contribute to local anesthetic toxicity.…”

“…Bupivacaine inhibited GIRK channels within seconds of application. In studies of other ion channels, bupivacaine inhibition took several minutes (4,5,46,47), leading to the proposition that the neutral form crosses the cell membrane and acts at an intracellular site. Lipophilic local anesthetics permeate the membrane very rapidly, however, as was shown for voltage-gated Na ϩ channels (48) and some K ϩ channels (6).…”

“…The inhibition of K ϩ channels, however, increases membrane excitability and could therefore contribute to bupivacaine-induced tachycardia and convulsions. Indeed, some local anesthetics inhibit voltage-gated K ϩ channels (4)(5)(6). The effect of local anesthetics on native inwardly rectifying K ϩ channels has been equivocal (5)(6)(7)(8).…”

The efficacy and safety of 0.75% levobupivacaine vs 0.75% bupivacaine for peribulbar anaesthesia

“…Further animal studies comparing the isomers of bupivacaine indicate a higher affinity and, therefore, a greater inhibition of the n-max with dextrobupivacaine than with levobupivacaine. 20 The dextro-enantiomer's greater affinity for myocardial potassium channels is even more marked, 21 and this may also be partially responsible for its cardiotoxicity. Furthermore, there is evidence that the local anaesthetic action on the cardiovascular centre of the brain may make an indirect contribution to the cardiovascular collapse.…”