Local anesthetic inhibits hyperpolarization-activated cationic currents

Abstract

Systemic administration of local anesthetics has beneficial perioperative properties and an anesthetic-sparing and antiarrhythmic effect, although the detailed mechanisms of these actions remain unclear. In the present study, we investigated the effects of a local anesthetic, lidocaine, on hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels that contribute to the pacemaker currents in rhythmically oscillating cells of the heart and brain. Voltage-clamp recordings were used to examine the properties of cloned HCN subunit currents expressed in Xenopus laevis oocytes and human embryonic kidney (HEK) 293 cells under control condition and lidocaine administration. Lidocaine inhibited HCN1, HCN2, HCN1-HCN2, and HCN4 channel currents at 100 μM in both oocytes and/or HEK 293 cells; it caused a decrease in both tonic and maximal current (∼30-50% inhibition) and slowed current activation kinetics for all subunits. In addition, lidocaine evoked a hyperpolarizing shift in half-activation voltage (ΔV(1/2) of ∼-10 to -14 mV), but only for HCN1 and HCN1-HCN2 channels. By fitting concentration-response data to logistic functions, we estimated half-maximal (EC(50)) concentrations of lidocaine of ∼30 to 40 μM for the shift in V(1/2) observed with HCN1 and HCN1-HCN2; for inhibition of current amplitude, calculated EC(50) values were ∼50 to 70 μM for HCN1, HCN2, and HCN1-HCN2 channels. A lidocaine metabolite, monoethylglycinexylidide (100 μM), had similar inhibitory actions on HCN channels. These results indicate that lidocaine potently inhibits HCN channel subunits in dose-dependent manner over a concentration range relevant for systemic application. The ability of local anesthetics to modulate I(h) in central neurons may contribute to central nervous system depression, whereas effects on I(f) in cardiac pacemaker cells may contribute to the antiarrhythmic and/or cardiovascular toxic action.

Fig. 1.

Local anesthetic lidocaine differentially inhibits HCN channel currents expressed in X. laevis oocytes. A, sample currents from X. laevis oocytes expressing mHCN1, mHCN2, and mHCN1-mHCN2 channel constructs evoked by hyperpolarizing voltage steps from −40 to −120 mV before and during exposure to lidocaine (100 μM); conditioning voltage steps were of different duration for the three constructs (3, 4, and 3 s) followed by a step to −90 mV for tail current analysis. B, summary data showing averaged (± S.E.M.) current inhibition (percentage from control; left) and shift in half-activation potential (V_1/2; right) evoked by lidocaine for each of the indicated HCN channel constructs. *, P < 0.05 by analysis of variance for lidocaine versus control (n = 6, 5, and 8 for mHCN1, mHCN2, and mHCN1-mHCN2, respectively).

Fig. 2.

Local anesthetic inhibits HCN channel currents in HEK 293 cells. A, sample currents from HEK 293 cells expressing mHCN1, mHCN2, or a linked heteromeric mHCN1-mHCN2 construct evoked by hyperpolarizing voltage steps (Δ −10 mV) from −40 to −120 mV before and during exposure to lidocaine (100 μM); voltage steps were followed by a step to −90 mV for tail current analysis. A sample current trace from untransfected HEK 293 cell is also shown at the bottom. B, activation curves were determined from tail currents (bottom) and steady-state I-V curves from currents at the end of the voltage steps (top) under control conditions (■), during exposure to lidocaine (▴) for mHCN1, mHCN2, or a linked mHCN1-HCN2 constructs. *, P < 0.05 versus control (n = 5, 5, and 6 for mHCN1, mHCN2, and mHCN1-HCN2, respectively).

Fig. 3.

Lidocaine inhibits HCN4 channel currents. A, sample currents from HEK 293 cells expressing mHCN4 channel construct evoked by hyperpolarizing voltage steps from −40 to −120 mV before and during exposure to lidocaine (100 μM); conditioning voltage steps (6–14 s) were followed by a step to −90 mV for tail current analysis. B, steady-state I-V curves from currents at the end of the voltage steps (top), and activation curves were determined from tail currents (bottom) under control conditions (■) and during exposure to lidocaine (▵). C. Summary data showing averaged (± S.E.M.) current inhibition (percentage from control; left) and shift in half-activation potential (V_1/2; right) evoked by lidocaine metabolite MEGX for each of the indicated HCN channel constructs. *, P < 0.05 versus control.

Fig. 4.

Lidocaine inhibits tonic HCN channel currents. A, instantaneous I-V relationships were obtained from a holding potential of −40 mV in HEK 293 cells expressing mHCN1, mHCN2, mHCN1-HCN2, and mHCN4 under control conditions (■), in the presence of 100 μM lidocaine (▴) and 3 mM CsCl (□). Solid lines represent linear fits through averaged data (± S.E.M.; n = 5, 5, 6, and 5 for HCN1, HCN2, HCN1-HCN2, and HCN4, respectively), representing the input conductance at −40 mV. B, input conductance was determined from slopes of instantaneous I-V curves in individual cells expressing the HCN channel constructs and averaged for each condition as indicated (C, control; L, lidocaine; Cs, CsCl). Lidocaine decreased input conductance in all HCN-expressing cells (*, P < 0.05 versus control).

Fig. 5.

Lidocaine causes a slowing of HCN subunit currents. Activation data were obtained from biexponential fits at −120 mV, and the time constant (τ) describing the fastest (and largest) current component were determined under control and during lidocaine application for mHCN1, mHCN1-mHCN2, mHCN2, and mHCN4 subunit currents under control conditions and in the presence of 100 μM lidocaine. Lidocaine caused a slowing of all HCN subunits current activation examined. *, P < 0.05 versus control (n = 5, 5, 6, and 5 for HCN1, HCN2, HCN1-HCN2, and HCN4, respectively).

Fig. 6.

Lidocaine modulates HCN channel currents at −70 mV and at clinically relevant concentrations. A, sample HCN1 current at −70 mV under control conditions and during administration of lidocaine (100 μM) and CsCl (3 mM), an HCN channel blocker. HCN1 currents include two components: a voltage- and time-dependent component that was almost strongly inhibited by lidocaine and totally blocked by Cs⁺, and a Cs⁺-sensitive instantaneous current component that was partly reduced by lidocaine. B, summary data showing effects of lidocaine on voltage- and time-dependent (left) and tonic (right) HCN currents measured at −70 mV in cells expressing HCN1, HCN2, HCN1-HCN2, and HCN4 channel constructs. Calculation of the percentage of inhibition of tonic current is relative to the Cs⁺-sensitive instantaneous current component (i.e., the HCN current). Lidocaine inhibited instantaneous and voltage-dependent currents for HCN1, HCN1-HCN2, and HCN4 channels; for HCN2, the instantaneous current is predominant at −70 mV and was also reduced by lidocaine. *, P < 0.05 versus control (n = 5, 5, 6, and 5 for HCN1, HCN2, HCN1-HCN2, and HCN4, respectively). C, averaged values for shift in amplitude inhibition (left) and V_1/2 (right) of mHCN1 (squares), mHCN2 (triangles), and heteromeric mHCN1-mHCN2 (circles) currents expressed in oocytes at different concentrations of lidocaine. The effects of lidocaine (0, 20, 50, 100, and 200 μM) on current amplitude and V_1/2 were averaged (± S.E.M.) and fitted with logistic equations.