Convergent and reciprocal modulation of a leak K+ current and I(h) by an inhalational anaesthetic and neurotransmitters in rat brainstem motoneurones

Abstract

Neurotransmitters and volatile anaesthetics have opposing effects on motoneuronal excitability which appear to reflect contrasting modulation of two types of subthreshold currents. Neurotransmitters increase motoneuronal excitability by inhibiting TWIK-related acid-sensitive K+ channels (TASK) and shifting activation of a hyperpolarization-activated cationic current (I(h)) to more depolarized potentials; on the other hand, anaesthetics decrease excitability by activating a TASK-like current and inducing a hyperpolarizing shift in I(h) activation. Here, we used whole-cell recording from motoneurones in brainstem slices to test if neurotransmitters (serotonin (5-HT) and noradrenaline (NA)) and an anaesthetic (halothane) indeed compete for modulation of the same ion channels - and we determined which prevails. When applied together under current clamp conditions, 5-HT reversed anaesthetic-induced membrane hyperpolarization and increased motoneuronal excitability. Under voltage clamp conditions, 5-HT and NA overcame most, but not all, of the halothane-induced current. When I(h) was blocked with ZD 7288, the neurotransmitters completely inhibited the K+ current activated by halothane; the halothane-sensitive neurotransmitter current reversed at the equilibrium potential for potassium (E(K)) and displayed properties expected of acid-sensitive, open-rectifier TASK channels. To characterize modulation of I(h) in relative isolation, effects of 5-HT and halothane were examined in acidified bath solutions that blocked TASK channels. Under these conditions, 5-HT and halothane each caused their characteristic shift in voltage-dependent gating of I(h). When tested concurrently, however, halothane decreased the neurotransmitter-induced depolarizing shift in I(h) activation. Thus, halothane and neurotransmitters converge on TASK and I(h) channels with opposite effects; transmitter action prevailed over anaesthetic effects on TASK channels, but not over effects on I(h). These data suggest that anaesthetic actions resulting from effects on either TASK or hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in motoneurones, and perhaps at other CNS sites, can be modulated by prevailing neurotransmitter tone.

Figure 1. Serotonin reverses halothane-induced membrane hyperpolarization in hypoglossal motoneurones

Effect of halothane (0.35 mm) and 5-HT (2 μM) on a hypoglossal motoneurone recorded under current clamp conditions. Halothane caused a membrane hyperpolarization that was reversed by 5-HT, even in the continued presence of halothane. The motoneurone was induced to fire by depolarizing DC injection (150 pA); action potentials were truncated by the chart recorder.

Figure 2. Serotonin and halothane elicit opposing actions on two distinct conductances in hypoglossal motoneurones

A, representative time series depicting the change in holding current (at −60 mV) induced by serotonin (5-HT, 5 μM); 5-HT caused an inward shift in holding current. B, sample current traces obtained at the times indicated in A in response to hyperpolarizing voltage steps from −60 mV under control conditions (a) and in the presence of 5-HT (b). Arrowheads indicate zero current level and open arrows indicate points used to determine instantaneous I-V relationships. A fixed step to −80 mV followed each test pulse (asterisks) in order to generate the tail currents that were used to derive the voltage dependence of I_h activation. C, the 5-HT-sensitive current was obtained by subtracting control currents (a) from those obtained in the presence of 5-HT (b). This current was associated with a decreased conductance but showed no clear reversal, suggesting modulation of multiple components. Inset, tail currents from control and 5-HT conditions were normalized, plotted with respect to the potential during the preceding step and fitted with a Boltzmann function to obtain the voltage dependence of I_h activation. 5-HT caused a depolarizing shift in I_h activation. D, time series illustrating the outward shift of holding current (at −60 mV) induced by halothane (0.9 mm) in a representative motoneurone. E, current traces obtained at the times indicated in D in response to hyperpolarizing voltage steps from −60 mV under control conditions (a) and in the presence of halothane (b). Note that tail currents are diminished in halothane (asterisks), reflecting decreased I_h amplitude. F, the halothane-sensitive current was obtained by subtracting control currents (a) from those obtained in the presence of halothane (b). This current was associated with an increased conductance, but with no clear reversal. Inset, normalized tail currents from control and halothane were fitted with a Boltzmann function; halothane caused a hyperpolarizing shift in the voltage dependence of I_h activation.

Figure 3. Halothane enhances net neurotransmitter-sensitive current

A, time series depicting the effect of halothane on 5-HT-sensitive current. Initially, 5-HT produced an inward shift in holding current (at −60 mV) that was reversible upon wash. Subsequently, halothane induced an outward current and in the continued presence of halothane, the 5-HT-induced inward current shift was enhanced. Note, however, that the absolute current level obtained with both 5-HT and halothane did not reach that observed with 5-HT alone (dashed line). B, averaged data depicting the magnitude of the 5-HT current. As compared with control, the 5-HT current was significantly enhanced in the presence of halothane. C, averaged I-V relationships of 5-HT-sensitive current obtained under control conditions (♦) and in the presence of halothane (▪). In the presence of halothane, the 5-HT current was associated with a greater reduction in conductance. D, mean data depicting the halothane-sensitive component of the 5-HT current. I-V data were derived by subtracting the 5-HT current in the presence of halothane from the control 5-HT current. Note that this current reverses ≈10 mV depolarized to E_K (arrowhead).

Figure 4. Pharmacological block of I_h revealed a TASK-like component of halothane- and neurotransmitter-sensitive current

A, when I_h was blocked by ZD 7288 (40 μM, pipette), the 5-HT-induced inward shift in current was enhanced by halothane; 5-HT inhibited all of the halothane-sensitive current (dashed line). B, averaged data depicting the enhanced 5-HT current amplitude in the presence of halothane. C, averaged I-V relationships of 5-HT-sensitive current obtained under control conditions (♦) and in the presence of halothane (▪). D, the halothane-sensitive component of 5-HT current, derived by subtraction of currents induced by 5-HT in the presence of halothane from those under control conditions, was well-fitted using the GHK constant field equation and reversed near E_K (arrowhead).

Figure 5. Effects of halothane on I_h are retained in acidified bath conditions that block motoneuronal anaesthetic- and neurotransmitter-sensitive K⁺ currents

A, the outward shift in holding current induced by halothane at pH 7.3 was reduced following bath acidification to block pH-sensitive K⁺ currents. B, averaged I-V data depict halothane-sensitive currents obtained at pH 7.3 (▪) and at pH 6.5 (♦); the anaesthetic-sensitive current under acidified conditions increased with hyperpolarization and was associated with a decreased conductance, consistent with a decrease in I_h. C, halothane produced a concentration-dependent hyperpolarizing shift in the voltage dependence of I_h activation. Data were obtained as described in Fig. 2. V_1/2 values (mV) were: control (pH 6.5), −101.9 ± 3.2; halothane (0.3 mm), −104. ± 3.4; halothane (0.4 mm), −107.6 ± 3.3; halothane (1.2 mm), −110.8 ± 3.6 (n = 8 for pH 6.5 and 0.3 mm halothane; n = 5 for 0.4 and 1.2 mm halothane). D, concentration-response curve for the halothane-induced shift in voltage dependence of I_h activation. The size of the halothane-induced shift in V_1/2 in cells exposed to multiple concentrations of halothane was plotted as a function of measured aqueous halothane concentrations. A least-squares fit of the data with a logistic equation predicted an EC₅₀ of 360 μM.

Figure 6. Halothane inhibits effects of 5-HT on I_h activation under acidified bath conditions

A, representative experiment in an acidified bath solution, when pH-sensitive K⁺ currents are blocked; under these conditions, halothane diminished the 5-HT-induced current. B, effect of 5-HT on the voltage dependence of I_h activation under control conditions and in the presence of halothane. Compared with control (♦), the V_1/2 of I_h activation was shifted to depolarized potentials by 5-HT (▪), but was shifted in the hyperpolarized direction by halothane (○), even when tested in the combined presence of 5-HT (□). Inset, the depolarizing shift in I_h activation produced by 5-HT was significantly reduced by halothane.