Effects of a quaternary bupivacaine derivative on delayed rectifier K(+) currents

Abstract

Block of hKv1.5 channels by R-bupivacaine has been attributed to the interaction of the charged form of the drug with an intracellular receptor. However, bupivacaine is present as a mixture of neutral and charged forms both extra- and intracellularly. We have studied the effects produced by the R(+) enantiomer of a quaternary bupivacaine derivative, N-methyl-bupivacaine, (RB(+)1C) on hKv1.5 channels stably expressed in Ltk(-) cells using the whole-cell configuration of the patch-clamp technique. When applied from the intracellular side of the membrane, RB(+)1C induced a time- and voltage-dependent block similar to that induced by R-bupivacaine. External application of 50 microM RB(+)1C reduced the current at +60 mV by 24+/-2% (n=10), but this block displayed neither time- nor voltage-dependence. External RB(+)1C partially relieved block induced by R-bupivacaine (61+/-2% vs 56+/-3%, n=4, P<0.05), but it did not relieve block induced by internal RB(+)1C. In addition, it did not induce use-dependent block, but when applied in combination with internal RB(+)1C a use-dependent block that increased with pulse duration was observed. These results indicate that RB(+)1C induces different effects on hKv1.5 channels when applied from the intra or the extracellular side of the membrane, suggesting that the actions of bupivacaine are the resulting of those induced on the external and the internal side of hKv1.5 channels.

Figure 1

Concentration-dependent effects of externally applied RB⁺1C (RB⁺1C_out) in hKv1.5 channels. (a) Original hKv1.5 current traces obtained in the absence and in the presence of RB⁺1C_out (50 and 500 μM) recorded after depolarizing the membrane potential from −80 mV to +60 mV during 250 ms. Deactivating tail currents were recorded after repolarizing to −40 mV. (b) Histogram representing the concentration dependent block induced by RB⁺1C_out. Columns represent the mean±s.e.mean of 3–10 experiments.

Figure 2

Effects of RB⁺1C_out on hKv1.5 channels. (a) Original records obtained in control conditions, in the presence of 50 μM RB⁺1C_out and after washout with drug-free external solution. Cells were held at −80 mV and 250 ms pulses every 10 s to membrane potentials between −80 and +60 mV in 10-mV steps, were applied. Depolarizing tail currents were recorded after repolarizing to −40 mV. (b) IV relationships obtained in the absence and in the presence of RB⁺1C_out. (c) Voltage dependence of block represented as the relative current at all membrane potentials studied. Individual data points represent the mean±s.e.mean of 10 experiments.

Figure 3

Effects of RB⁺1C_out (50 μM) in hKv1.5 pore mutant channels located at the P-loop (T477S) and the S6 segment (T505A and V512M). Current traces were elicited after applying 250 ms depolarizing pulses from a holding potential of −80 mV (T477S and T505A) or −100 mV (V512M) to +60 mV. Tail currents were recorded at −40 mV (T477S and T505A) or at −60 mV (V512M).

Figure 4

Effects of RB⁺1C_out on Kv2.1 channels. (a) Original records obtained in control conditions and in the presence of 50 μM RB⁺1C_out. (b) IV relationships obtained in the absence and in the presence of RB⁺1C_out. The dotted line represents the zero level. (c) Voltage dependence of block represented as the relative current at all membrane potentials studied. The dotted line represents the activation curve and the solid line is the Boltzmann fit to data points positive to 0 mV. Individual data points represent the mean±s.e.mean of eight experiments.

Figure 5

Effects of internally applied RB⁺1C (RB⁺1C_in) in hKv1.5 channels. In these experiments, the pipette was filled with an internal solution containing RB⁺1C and the tip of the pipette was filled with drug-free internal solution. (a) Development of block induced by RB⁺1C_in (500 μM) during the first 15 min after rupture of the seal membrane, which was taken as time 0′. (b) Original hKv1.5 current traces obtained in the absence and in the presence of RB⁺1C_in at 100 μM and 500 μM. (c) Histogram representing the concentration dependent block induced by RB⁺1C_in. Columns represent the mean±s.e.mean of 5–12 experiments.

Figure 6

Effects of RB⁺1C_in on hKv1.5 channels. (a) Original records obtained just after breaking the seal (‘control') and when the steady state block induced by 500 μM RB⁺1C_in was achieved (∼15′). (b) IV relationships under ‘control' conditions and after 15′ of dialysis with RB⁺1C-containing internal solution. (c) Voltage dependence of block, plotted as the relative current at all membrane potentials studied. This voltage dependence was consistent with a fractional electrical distance (δ) of 0.34±0.04. Individual data points represent the mean±s.e.mean of six experiments.

Figure 7

Competition between R-bupivacaine and RB⁺1C_out (left panels) and effects of RB⁺1C_out in cells dialyzed with RB⁺1C_in (right panels). (a) Original current records elicited by the application of a depolarizing pulse from −80 to +60 mV in the presence of R-bupivacaine (5 μM) alone and in the presence of the combination of R-bupivacaine plus RB⁺1C_out (50 μM). (b) The IV relationship obtained under control conditions, in the presence of R-bupivacaine and in the presence of the combination of R-bupivacaine plus RB⁺1C_out. Individual data points represent the mean±s.e.mean of four experiments. (c) Original records obtained in the presence of 20 μM RB⁺1C_in alone and in the presence of RB⁺1C_in plus RB⁺1C_out. (d) Shows the IV relationship obtained in cells internally perfused with RB⁺1C_in in the absence and in the presence of RB⁺1C_out. Individual data points represent the mean±s.e.mean of 10 experiments.

Figure 8

Lack of use-dependent effects of RB⁺1C_out in hKv1.5 channels. (a) Normalized outward hKv1.5 peak current amplitude in control conditions and in the presence of RB⁺1C_out (50 μM) during trains of 15 pulses of 250 ms and 1000 ms in duration applied from −80 to +50 mV in which the depolarizing steps were separated from each other by a fixed interstimulus interval of 1000 ms. (b) Relative current (I_Drug/I_Control) plotted as a function of the number of pulses in the train. (c) Recovery process of hKv1.5 current under control conditions and in the presence of RB⁺1C_out. The maximum peak amplitude of outward hKv1.5 currents elicited by a test pulse was plotted as a function of the interstimulus interval in the absence and presence of RB⁺1C_out. Continuous lines represent the best biexponential fit of the increase of hKv1.5 currents as a function of the interstimulus interval from which the recovery time constants were obtained. Individual data points represent the mean±s.e.mean of five experiments.

Figure 9

Use-dependent effects of RB⁺1C_out in hKv1.5 channels in cells dialyzed with RB⁺1C_in. (a) Outward hKv1.5 peak current amplitude in the presence of RB⁺1C_in (20 μM) alone and the combination of RB⁺1C_in plus RB⁺1C_out (50 μM) when applying trains of 15 pulses of 250 and 1000 ms in duration applied from −80 to +50 mV in which depolarizing steps were separated each other by a fixed interstimulus interval of 1000 ms. (b) Relative current plotted as a function of the number of pulses in the train. (c) Recovery process of hKv1.5 current in the presence of RB⁺1C_in and of RB⁺1C_in plus RB⁺1C_out. The maximum peak amplitude of outward hKv1.5 currents elicited by a test pulse was plotted as a function of the interstimulus interval in both experimental conditions. Continuous lines represent the best biexponential fit of the increase of hKv1.5 currents as a function of the interstimulus interval from which the time constants of recovery were obtained. Individual data points represent the mean±s.e.mean of five experiments.