Stereoselective effects of the enantiomers of a new local anaesthetic, IQB-9302, on a human cardiac potassium channel (Kv1.5)

Abstract

1. The N-substituent of IQB-9302 has the same number of carbons as bupivacaine, but it exhibits a different spatial localization (n-butyl vs cyclopropylmethyl). Thus, the study of the effects of IQB-9302 enantiomers on hKv1.5 channels will lead to a better knowledge of the determinants of stereoselective block. 2. The effects of the IQB-9302 enantiomers were studied on hKv1.5 channels stably expressed in LTK:(-) cells using the whole-cell configuration of the patch-clamp technique. Drug molecular modelling was performed using Hyperchem software. 3. Block induced by IQB-9302 was stereoselective with the R(+) enantiomer being 3.2-fold more potent than the S(-) one (K(D) of 17.8+/-0.5 microM vs 58.6+/-4.0 microM). 4. S(-)- and R(+)IQB-9302 induced-block was time- and voltage-dependent consistent with an electrical distance from the cytoplasmic side of 0.173+/-0.022 (n=12) and 0.181+/-0.018 (n=10), respectively. 5. Potency of block of pipecoloxylidide local anaesthetics was linearly related to the length between the cationic tertiary amine and the end of the substituent. 6. Molecular modelling shows that only when S(-) and R(+) enantiomers are superimposed by their aromatic ring, their N-substituents are in opposite directions, which can explain the stereospecific block induced by bupivacaine and IQB-9302 with hKv1.5 channels. 7. These results suggest that: (a) IQB-9302 enantiomers block the open state of hKv1.5 channels, and (b) the length of the N-substituent in these local anaesthetics and not its volume determines the potency and degree of their stereoselective hKv1.5 channel block.

Figure 1

Chemical structure of bupivacaine and IQB-9302. The asterisk indicates the asymmetric carbon in the molecule.

Figure 2

Effects of IQB-9302 enantiomers on hKv1.5 currents. Currents are shown for depolarization from −80 mV to voltages between −80 and +60 mV in steps of 10 mV. Tail currents were obtained on return to −40 mV. Traces were obtained in the absence (left panel), in the presence of 50 μM of each enantiomer of IQB-9302 (middle panel) and after washout with drug-free external solution (right panel). All records shown in this Figure were recorded from the same cell, which was first perfused with S(−)IQB-9302 and afterwards with R(+)IQB-9302. The control for R(+)IQB-9302 is the washout of S(−)IQB-9302. Cell capacitance, 23 pF.

Figure 3

Concentration dependence of IQB-9302 enantiomers-induced block of hKv1.5 channels. Reduction of current (relative to control) at the end of depolarizing steps from −80 mV to +60 mV was used as index of block. Data are mean±s.e.mean of a total of 51 experiments. The continuous line represents the fit of the experimental data to the equation: 1/[1+(K_D/[D])ⁿ,_H].

Figure 4

Voltage dependence of hKv1.5 block by IQB-9302 enantiomers. (A) Current-voltage relationship (250 ms isochronal) in control conditions and in the presence of 50 μM S(−)IQB-9302. (B) Relative current expressed as I_{S(−)IQB-9302}/I_Control from data shown in (A). (C) Current-voltage relationship (250 ms isochronal) in control conditions and in the presence of 10 μM R(+)IQB-9302. (D) Relative current expressed as I_R(+)IQB-9302/I_Control from data shown in (C). The dashed lines in (B) and (D) represent the activation curve of the hKv1.5 channel for each experiment. In both cases, block increased steeply between −20 and 0 mV, which corresponds to the voltage range of activation of hKv1.5. For membrane potentials positive to 0 mV, a continued but more shallow voltage dependence was observed. This voltage dependence was fitted (continuous line) with eq. 4 (see Methods) and yielded δ∼0.18.

Figure 5

Time-dependent block induced by IQB-9302 enantiomers. (A) Superimposed traces for steps from −80 mV to +60 mV under control conditions and in the presence of 500 μM S(−)IQB-9302 and 50 μM R(+)IQB-9302. (B) Tail current crossover. Currents recorded in control conditions and in the presence of 20 μM S(−)IQB-9302 or 10 μM R(+)IQB-9302 were superimposed. Tail currents were obtained at −40 mV after a 250 ms-depolarizing pulse to +60 mV. Arrow shows the crossover of tracings recorded in the presence of IQB-9302 enantiomers with those recorded under control conditions.

Figure 6

Relationship between the potency of block of hKv1.5 channels and the maximal length between the tertiary amine and the end of the N-substituent in mepivacaine, IQB-9302, ropivacaine and bupivacaine.

Figure 7

Docking of minimum energy conformers of the two enantiomers of bupivacaine and IQB-9302 superimposed by the pipecoloxylidide ring (A,B) or by the aromatic ring (C,D). In both cases, the R(+) enantiomers are shown with thicker lines than the S(−) ones. Note that only when each pair of enantiomers are superimposed by the pipecoloxylidide a clear difference in the spatial location of the drugs is observed. This finding could explain the experimental differences in potency observed for R(+) and S(−) enantiomers. Both models were constructed by using the Hiperchem program.