Bupivacaine effects on hKv1.5 channels are dependent on extracellular pH

Abstract

1. Bupivacaine-induced cardiotoxicity increases in hypoxic and acidotic conditions. We have analysed the effects of R(+)bupivacaine on hKv1.5 channels stably expressed in Ltk(-) cells using the whole-cell patch-clamp technique, at three different extracellular pH (pH(o)), 6.5, 7.4 and 10.0. 2. Acidification of the pH(o) from 7.4 to 6.5 decreased 4 fold the potency of R(+)bupivacaine to block hKv1.5 channels. At pH(o) 10.0, the potency of the drug increased approximately 2.5 fold. 3. Block induced by R(+)bupivacaine at pH(o) 6.5, 7.4 and 10.0, was voltage- and time-dependent in a manner consistent with an open state block of hKv1.5 channels. 4. At pH(o) 6.5, but not at pH(o) 7.4 or 10.0, R(+)bupivacaine increased by 95+/-3 % (n=6; P<0.05) the hKv1.5 current recorded at -10 mV, likely due to a drug-induced shift of the midpoint of activation (DeltaV=-8.5+/-1.4 mV; n=7). 5. R(+)bupivacaine development of block exhibited an 'instantaneous' component of block at the beginning of the depolarizing pulse, which averaged 12.5+/-1.8% (n=5) and 4.6+/-1.6% (n=6), at pH(o) 6.5 and 7.4, respectively, and that was not observed at pH(o) 10.0. 6. It is concluded that: (a) alkalinization of the pH(o) increases the potency of block of R(+)bupivacaine, and (b) at pH(o) 6.5, R(+)bupivacaine induces an 'agonist effect' of hKv1.5 current when recorded at negative membrane potentials.

Figure 1

Effects of R(+)bupivacaine on hKv1.5 channels at different pH_o values. Original records obtained in the absence and in the presence of equipotent drug concentrations after applying depolarizing pulses from −80 to +60 mV in 10 mV steps of 250 ms duration. Tail currents were recorded upon repolarization to −40 mV (pH_o 7.5 and 10.0) or −30 mV (pH_o 6.5). (A) Shows original records obtained at pH_o 6.5, 7.4 and 10.0 in the absence and in the presence of equipotent concentrations of R(+)bupivacaine. R(+)bupivacaine 20, 5 and 1 μM induced a time-dependent block which yields 62±4% (n=6), 61±2% (n=5) and 47±5% (n=5) at pH_o 6.5, 7.4 and 10.0, respectively. (B) Shows the activation curves at pH_o 6.5, 7.4 and 10.0 in the absence and in the presence of equipotent R(+)bupivacaine concentrations. The midpoint of activation (E_h) was shifted to more negative potentials as the pH_o value was more alkaline. Data points represent the mean±s.e.mean of a 6 – 7 experiments. ^*P<0.01.

Figure 2

Effects of changes in extracellular pH (pH_o) on hKv1.5 channels. Considering the equation ΔE_h=A/(1+10^(pH-pKa)); where A represents the maximum amplitude, and assuming that E_h saturates at pH_o 10.0 (so ΔE_h=0 at pH_o=10.0), we calculated a pKa value of 6.44. This is in close agreement with the pKa value of protonation for an histidine (6.0) and suggests that the titration of a histidine present within the pore region of hKv1.5 channels could be responsible for the observed proton-induced shift towards more positive membrane potentials as the pH_o is reduced.

Figure 3

Concentration-response curves for R(+)bupivacaine-induced block at all the pH_o studied. In this plot we represented the R(+)bupivacaine induced block at each concentration and under each experimental conditions. At pH_o 6.5 and 10.0 the concentration-response curves were better fit assuming a Hill equation of two components, i.e. biphasic concentration-response curves, from which two K_D values were obtained (K_D1 and K_D2). Dotted lines represent the concentration-response curves when the fit assumed a Hill equation of one component. The concentration-response curve for R(+)bupivacaine at pH_o 7.4 (Δ) was taken from (Valenzuela et al., 1995a) with permission.

Figure 4

Voltage-dependent effects of equipotent R(+)bupivacaine concentrations on hKv1.5 channels at different pH_o values. Relationship between the relative current (I_Drug/I_Control) and membrane potential at pH_o 6.5, 7.4 and 10.0. At the three pH_o studied, block induced by R(+)bupivacaine steeply increased in the range of activation of the channel. At voltages positive to +10 mV (pH_o 6.5), 0 mV (pH_o 7.4) and −5 mV (pH_o 10.0) block increased with a shallower voltage-dependence consistent with δ values of 0.18±0.01 (n=14), 0.18±0.01 (n=5) and 0.16±0.01 (n=7), respectively (P>0.05).

Figure 5

Effects of R(+)bupivacaine on hKv1.5 channels at pH_o 6.5. Left panel shows original records obtained after depolarizing the cell from −80 to +60 mV. Right panel shows the drug effects after depolarizing the cell membrane to −10 mV. Note that, under these circumstances, the drug increased the amplitude of the hKv1.5 current by 95±3% (n=6; P<0.05), whereas after strong depolarizations (left panel), R(+)bupivacaine inhibited the hKv1.5 current.

Figure 6

Time-dependent block of hKv1.5 channels induced by R(+)bupivacaine at pH_o 6.5, 7.5 and 10.0. (A) Shows original records obtained after applying a depolarizing pulse from −80 mV to +60 mV for 250 ms in duration in the absence and in the presence of R(+)bupivacaine (20, 5 and 1 μM) at the different pH_o values. R(+)bupivacaine induced a fast initial decline of the current which was faster at higher drug concentrations and thus, it was considered as a good index of the time constant of block (τ_Block). (B) The inverse of τ_Block was plotted versus bupivacaine concentration. For a first-order blocking scheme, a linear relation is expected: τ_Block⁻¹=k×[D]+l (see Methods section). The solid line represents the linear fit, from which the apparent binding and unbinding rate constants were obtained. Inset: Representative traces of time course of the development of block of hKv1.5 after a depolarizing pulse to +60 mV from a holding potential of −80 mV at pH_o 6.5, 7.4 and 10.0 induced by R(+)bupivacaine at concentrations of 10, 5 and 1 μM, respectively. The reduction of hKv1.5 current in the presence of the drug is expressed as a proportion of the control current at any given time after the beginning of the depolarizing pulse. In the presence of R(+)bupivacaine at all the pH_o studied inhibition of the current increases exponentially during depolarization. Note that at pH_o 6.5 and 7.4, R(+)bupivacaine (10 μM) induced an instantaneous block at t=0 ms.

Figure 7

Time-dependent block of hKv1.5 channels induced by R(+)bupivacaine at different pH_o values observed during deactivation at −40 mV (at pH_o 7.4 and 10.0) and −30 mV (at pH_o 6.5). R(+)bupivacaine induced an initial rising phase due to the unblocking of R(+)bupivacaine from open channels and slowed the deactivation time course at all the pH_o studied which resulted in a crossover phenomenon indicative of an open-channel block mechanism. The arrows indicate the ‘crossover' between the tail currents recorded in the presence of drug and those recorded under control conditions.