Cation and voltage dependence of lidocaine inhibition of the hyperpolarization-activated cyclic nucleotide-gated HCN1 channel

Abstract

Lidocaine is known to inhibit the hyperpolarization-activated mixed cation current (I_h) in cardiac myocytes and neurons, as well in cells transfected with cloned Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels. However, the molecular mechanism of I_h inhibition by this drug has been limitedly explored. Here, we show that inhibition of I_h by lidocaine, recorded from Chinese hamster ovary (CHO) cells expressing the HCN1 channel, reached a steady state within one minute and was reversible. Lidocaine inhibition of I_h was greater at less negative voltages and smaller current amplitudes whereas the voltage-dependence of I_h activation was unchanged. Lidocaine inhibition of I_h measured at -130 mV (a voltage at which I_h is fully activated) was reduced, and I_h amplitude was increased, when the concentration of extracellular potassium was raised to 60 mM from 5.4 mM. By contrast, neither I_h inhibition by the drug nor I_h amplitude at +30 mV (following a test voltage-pulse to -130 mV) were affected by this rise in extracellular potassium. Together, these data indicate that lidocaine inhibition of I_h involves a mechanism which is antagonized by hyperpolarizing voltages and current flow.

Figure 1

Lidocaine inhibits HCN1-mediated I_h conductance to a greater extent at less negative voltages but does not alter the voltage-dependence of I_h activation. (A) Representative traces of I_h activated by membrane voltages in the range from −30 mV to −135 mV at 30 mM extracellular potassium under control conditions and following application of 600 µM lidocaine. Activation voltage pulses were followed by a repolarization to −65 mV. (B) Plots of steady-state I_h, before and after lidocaine, versus voltage, collected from current traces as shown in ‘A’; I_h amplitudes in the presence of lidocaine were normalized to those in the control solution lacking lidocaine. (C) Plots of G_h, determined from tail current amplitudes at −65 mV as shown in ‘A’, before and after lidocaine, versus voltage; I_h amplitudes in the presence of lidocaine were normalized to those in the control solution lacking lidocaine. The solid curved line represents fitting of the values by a single exponential Boltzmann relation. Neither the mid-activation voltage (V _1/2) of G_h nor the slope factor of this relationship was altered (n = 4; −87.2 ± 2.7 vs. −86.9 ± 3.6 mV and 8.5 ± 0.6 vs. 8.7 ± 0.6; paired t-test, P = 0.79 and P = 0.89, respectively). (D) Plots of the fraction of steady-state I_h and G_h inhibited by lidocaine versus voltage. Steady-state I_h inhibition increased from 43.9 ± 5.8% at −135 to 62.4 ± 3.3% at −90 mV (n = 4; ANOVA, P = 0.038) whereas G_h was not altered significantly (n = 4; 69.3 ± 2.0 versus 70.5 ± 2.1%; P = 0.98).

Figure 2

Raising extracellular potassium increases I_h amplitude at −130 mV in CHO cells that express the HCN1 isoform. (A) Representative traces of I_h elicited by the voltage protocol shown below them from the same cell exposed to extracellular potassium concentrations of 5.4 and 60 mM. The magnitude of I_h (for both inward current determined at −130 mV and outward current determined at +30 mV) was calculated as the difference between the instantaneous current at the beginning of each pulse (I_inst) and the steady-state current (I_ss) at the end of the pulse (arrows). Note the large difference in inward current measured at −130 mV and very little difference in current measured at +30 mV between the two extracellular solutions. (B) Amplitudes of I_h density (nA/pF) measured at −130 mV (inward current) and +30 mV (outward current), plotted as a function of the extracellular potassium concentration. Increasing extracellular K⁺ from 5.4 mM to 30 mM and to 60 mM produced a concentration-dependent increase in the average amplitude inward I_h, which was measured at the end of the voltage pulse to −130 mV (filled circles; n = 22–32; ANOVA, P < 0.0001). The curved line represents a fit of the current density from individual cells to a Michaelis-Menten equation, yielding an apparent dissociation constant of 13.8 ± 7.1 mM. Maximum outward “tail” I_h, which was measured 1 ms after the voltage step to +30 mV to minimize the contribution of capacitance current, was not significantly affected by raising the concentration of extracellular potassium (filled squares; P = 0.12). The reversal potentials for I_h in different concentrations of extracellular potassium and sodium were −26 ± 5 mV (5.4 mM Na, 135 mM K), −20 ± 4 mV (30 mM K, 110 mM Na), and −5 ± 9 mV (60 mM K, 80 mM Na); extracellular sodium concentrations were reduced proportionally to maintain osmolarity (see Materials and Methods).

Figure 3

Raising extracellular potassium increases the rate of I_h deactivation without significantly affecting the voltage dependence and rate of I_h activation. (A) Representative traces of I_h activated by the voltage protocol shown below the traces at 5.4 mM, 30 mM, and 60 mM potassium in the extracellular recording solution. (B) Plots of normalized tail current amplitudes at −65 mV (G_h) versus test voltage at the three concentrations of extracellular potassium. Curved lines represent fits to a Boltzmann function (cf. Material and Methods). Mid-activation voltages (V½) are −79.2 ± 3.6 mV, −82.2 ± 2.3 mV, and −84.7 ± 3.9 mV, at 5.4, 30, and 60 mM extracellular potassium, respectively, and were not significantly different (n = 7–11; ANOVA, P = 0.52). The corresponding values for slope factor (k) were 9.6 ± 0.5, 10.5 ± 0.7 and 12.4 ± 0.8, and were significantly different (n = 7–11; ANOVA, P = 0.045). (C) Plots of fast (τ _fast) and slow (τ _slow) time constants of activation of steady-state I_h measured at −120 mV as a function of extracellular potassium concentration. The values obtained at different potassium concentrations were not significantly different (n = 7–11; ANOVA, P = 0.42 (τ _fast), P = 0.3 (τ _slow)). (D) Plot of time constants of I_h deactivation as a function of the extracellular potassium concentration. The values of time constants of I_h deactivation was significantly different between values obtained at 5.4 mM, 30 mM and 60 mM extracellular potassium (n = 5–11; ANOVA, P < 0.001).

Figure 4

Elevation of extracellular potassium concentration opposes the effects of lidocaine on HCN1-mediated I_h amplitude. (A) Representative traces of inward steady-state and outward I_h under control conditions and following application of 100 µM lidocaine with a concentration of 5.4 mM, 30 mM, or 60 mM of potassium in the extracellular solution. Activation voltage pulses (1 s) producing inward steady-state current, followed by by a repolarization pulse to +30 mV to generate outward current, were applied with an interval of 15 s (B). Amplitudes of steady-state inward I_h (−130 mV) and maximum outward I_h (+30 mV) from individual cells with 5.4 mM, 30 mM, or 60 mM potassium in the extracellular solution under control conditions and in the presence of lidocaine (100 µM). The horizontal bar denotes the duration of lidocaine exposure. Note that the inhibition of inward and outward I_h by lidocaine was reversible. (C) Bar graphs of percent inhibition of I_h (left, −130 mV; right, +30 mV) by 100 mM lidocaine at different concentrations of extracellular potassium. Lidocaine produced reversible inhibition of I_h at −130 mV (inward) or +30 mV (maximum outward current) by 48.2 ± 3.4 and 30.2 ± 2.1%, respectively, (n = 6; paired t-test, P < 0.001) at 5.4 mM extracellular potassium. The percentage of I_h inhibited by this concentration of lidocaine was less at 30 and 60 mM extracellular potassium (n = 4–6; ANOVA, P < 0.001 at both voltages; n = 4–6 cells for each concentration).

Figure 5

The concentration-response curve for the effects lidocaine and extracellular potassium on HCN1-mediated I_h at −130 mV demonstrates changes that are consistent with an interaction between them. (A) Plots of I_h amplitude at −130 mV versus concentration of extracellular potassium concentration in the presence of given concentrations of lidocaine (100, 200, and 600 µM). The solid lines represent fits to the Michaelis-Menten equation. An increase in the concentration of lidocaine from 100 to 600 µM resulted in an increase in the apparent dissociation constant of potassium from 5.3 ± 0.5 to 27.3 ± 8.9 mM (n = 4–6; ANOVA, P < 0.0001). (B) Plots of percent I_h inhibition at −130 mV versus lidocaine concentration. The solid line represents a fit using the Hill equation, which yielded values for IC₅₀ of 78 ± 7, 495 ± 133, and 332 ± 151 µM (n = 4–6 cells; ANOVA, P < 0.0001) and estimated Hill coefficients of 1.6 ± 0.2, 0.84 ± 0.12, and 0.92 ± 0.3 at 5.4, 30, and 60 mM extracellular potassium, respectively. Lidocaine’s estimated maximum inhibition was also lower at the higher concentrations of extracellular potassium, declining from 80.7 ± 3.5 to 71.1 ± 5.9% and to 26.6 ± 3.4% upon raising the extracellular potassium concentration from 5.4 to 30 mM, and to 60 mM, respectively.

Figure 6

Lidocaine inhibition of HCN1-mediated I_h is unaltered at +30 mV by raising the concentration of extracellular potassium (A). Representative traces of outward tail I_h relaxation, recorded at +30 mV following hyperpolarization to −130 mV from a holding voltage of −35 mV, under control conditions and following application of 100 µM lidocaine, at 30 mM extracellular potassium. The tail currents are shown after 1 ms in order to remove any contribution from the capacitive transient and clarity. (B) Mean time courses of lidocaine (600 µM) inhibition of the outward I_h at different concentration of extracellular potassium (5.4, 30, and 60 mM) obtained by normalizing tail currents in the presence of lidocaine from those recorded in control solutions as shown in ‘A’. Time courses were fit with double exponential equations. The extent of inhibition at the beginning of the voltage pulse to +30 mV increased over the course of deactivation for each concentration of extracellular potassium. The initial value for inhibition was larger for the lower concentrations of extracellular potassium, and they reflected the mean values plotted in Fig. 4C. Maximum inhibition reached a plateau and was the same at all potassium concentrations (~70–80%). Raising extracellular potassium from 5.4 to 60 mM did not significantly alter either the fast or slow time constants of lidocaine block, which varied from 1.1 ± 0.13 to 1.33 ± 0.15 ms and from 8.1 ± 0.25 to 9.6 ± 0.9 ms (n = 4–5; ANOVA, P = 0.66 and 0.51), respectively, in the presence of 600 µM lidocaine. (C) Plots of normalized charge transfer versus lidocaine concentration at different extracellular potassium concentrations (5.4, 30, and 60 mM). To estimate the total charge transferred, the area under leak subtracted traces of tail I_h relaxation was calculated by integration and those calculated in the presence of lidocaine were normalized to those calculated in the absence of lidocaine. The solid line represents a fit using the Hill equation, which produced IC₅₀ values of 56 ± 12, 84 ± 8, and 56 ± 7 µM (n = 4–6; ANOVA, P < 0.0001), Hill coefficients of 1.3 ± 0.3, 0.98 ± 0.11, and 1.49 ± 0.23, and maximum inhibitions of 82.2 ± 6.9, 88.1 ± 2.4, and 71.7 ± 2.4%, 5.4, 30, and 60 mM extracellular potassium, respectively. Values shown are those collected from individual experiments.