Enhancement of delayed-rectifier potassium conductance by low concentrations of local anaesthetics in spinal sensory neurones

Abstract

Combining the patch-clamp recordings in slice preparation with the 'entire soma isolation' method we studied action of several local anaesthetics on delayed-rectifier K(+) currents in spinal dorsal horn neurones. Bupivacaine, lidocaine and mepivacaine at low concentrations (1 - 100 microM) enhanced delayed-rectifier K(+) current in intact neurones within the spinal cord slice, while exhibiting a partial blocking effect at higher concentrations (>100 microM). In isolated somata 0.1 - 10 microM bupivacaine enhanced delayed-rectifier K(+) current by shifting its steady-state activation characteristic and the voltage-dependence of the activation time constant to more negative potentials by 10 - 20 mV. Detailed analysis has revealed that bupivacaine also increased the maximum delayed-rectifier K(+) conductance by changing the open probability, rather than the unitary conductance, of the channel. It is concluded that local anaesthetics show a dual effect on delayed-rectifier K(+) currents by potentiating them at low concentrations and partially suppressing at high concentrations. The phenomenon observed demonstrated the complex action of local anaesthetics during spinal and epidural anaesthesia, which is not restricted to a suppression of Na(+) conductance only.

Figure 1

Potentiation of delayed-rectifier K⁺ current by local anaesthetics in intact spinal sensory neurones. (A) Delayed-rectifier K⁺ currents activated by a voltage pulse to +20 mV following a 150 ms prepulse to −60 mV in control solution and in the presence of 10 and 300 μM bupivacaine, lidocaine and mepivacaine. Holding potential −80 mV. (B) Relative amplitudes of the currents at +20 mV as a function of bupivacaine (circles), lidocaine (triangles) and mepivacaine (squares) concentration. The currents were normalized by the amplitude of the corresponding current recorded in control solution. Each data point is the mean of measurements from five cells. Connecting line was drawn by eye.

Figure 2

Effect of bupivacaine on the amplitude of the delayed-rectifier K⁺ current in isolated somata. (A) Delayed-rectifier currents activated by depolarization to +20 mV following a 150 ms prepulse to −60 mV in the presence of different concentrations of bupivacaine (indicated near the corresponding trace). Holding potential, −80 mV. (B) Amplitudes of delayed-rectifier K⁺ currents activated by different depolarizing potentials as a function of bupivacaine concentration. Each data point represents the mean of five measurements in different somata. All amplitudes were normalized by the amplitude of the control current recorded at +40 mV (I_control(+40 mV)). The data for −50 and −40 mV are shown at higher resolution. Data points are connected by eye.

Figure 3

Effect of bupivacaine on delayed-rectifier K⁺ conductance in isolated somata. (A) Voltage-dependence of delayed-rectifier K⁺ conductance studied at different bupivacaine concentrations. Reversal potential for K⁺ ions was assumed to be −84 mV. Data from five somata. Data points were fitted with a standard Boltzmann function and normalized by the maximum conductance measured in control solution: where G is conductance, G_M is the maximal conductance at a given concentration of blocker, G_M(0) is maximal conductance in control solution, E is membrane potential, E₅₀ is a potential at which a half-maximal conductance is activated and k is steepness factor. The fitting parameters are given in Table 1. (B) The same characteristics as in A normalized to 1.

Figure 4

Acceleration of the current activation by bupivacaine. (A) delayed-rectifier K⁺ currents in the presence of different bupivacaine concentrations. The currents were activated using the same pulse protocol as in Figure 2A. Beginning of the depolarizing pulse is indicated by an arrowhead. (B) Current traces normalized to 1. (C) Time constant of half-maximal activation (τ_0.5) as a function of membrane potential in control solution and in the presence of 1 μM and 1 mM bupivacaine. Data from six somata. The data points were fitted with a mono-exponential function: A×exp[−(E−ΔE)/k]. For the data in control solution ΔE was assumed to be zero and the values of A=5.4±0.1 ms and k=31.8±0.8 mV were obtained by optimal fitting (r=0.999). For 1 μM and 1 mM bupivacaine, the values of A and k were fixed at 5.4 ms and 31.8 mV, respectively, and the ΔE parameter was varied to give best fit −13.1±0.5 mV (r=0.999) for 1 μM bupivacaine and −21.3±0.7 mV (r=0.997) for 1 mM bupivacaine.

Figure 5

Control experiments with Na⁺ currents. (A) Na⁺ currents activated in an isolated soma by different depolarizing pulses (indicated near the corresponding trace) in control solution and in 1 μM bupivacaine. Holding potential, −80 mV. Below, the current-voltage relationships for Na⁺ currents in control solution (open symbols) and in the presence of 1 μM bupivacaine (filled symbols). Connection lines were drawn by eye. (B) Voltage-dependence of Na⁺ conductance in control solution (circles) and in the presence of 1 μM bupivacaine (stars). The reversal potential for Na⁺ ions was assumed to be +53 mV. The data points (five somata) were fitted using the Boltzmann function and normalized to 1. Fitting parameters are given in the text.

Figure 6

Bupivacaine increases the P_o but does not change the amplitude of the single-channel current. (A) Recordings from an inside-out patch containing only delayed-rectifier K⁺ channels (at least two active) before and after addition of 1 μM bupivacaine to bath solution. Holding potential, −80 mV. The channels were activated by a depolarization to +40 mV following a 250 ms prepulse to −60 mV. The lowermost traces are averages of total 70 episodes each. Note that the averaged traces are given at higher resolution. The averaged currents were smaller than the single-channel currents because some episodes either were empty or had only a few channel openings. (B) All point-amplitude histograms, each based on all 70 episodes. The histograms are fitted using three Gauss functions with the amplitudes A₀=4171 (σ=0.187 pA), A₁=698 (σ=0.356 pA; i₁=1.01 pA) and A₂=19 (σ=0.167 pA; i₂=2.20 pA) for control solution and A₀=3620 (σ=0.182 pA), A₁=1172 (σ=0.383 pA; i₁=1.05 pA) and A₂=89 (σ=0.361 pA; i₂=2.31 pA) for 1 μM bupivacaine. Using the equation given in the methods section, the P_o was calculated to be 0.075 for control solution and 0.14 for 1 μM bupivacaine. (C) All point-amplitude histogram for delayed-rectifier channels in the presence and absence of 1 μM bupivacaine. For these histograms, only the episodes with very clear openings were selected (10 of total 70 for each solution). In addition, some parts of the recording at the level of the base line were digitally ‘cut out', to increase the relative amplitude of the peak corresponding to the channel opening. The data were fitted using the sum of three Gauss functions as described in the method section. The single-channel currents determined from the fitting were i₁=1.08±0.01 pA in control solution and i₁=1.11±0.01 pA in 1 μM bupivacaine.

Figure 7

Block of K⁺ channels by high concentrations of bupivacaine. Single K⁺ channel currents in control solution and in the presence of 100 μM bupivacaine. The experimental protocol was the same as in Figure 6A. Current traces were digitally filtered at 200 Hz.