TASK-like K+ channels mediate effects of 5-HT and extracellular pH in rat dorsal vagal neurones in vitro

Abstract

Dorsal vagal neurones (DVN) receive serotonergic projections from the medullary raphé nuclei, suggesting that 5-HT modulates vagal activity. A previous study has shown that 5-HT excites DVN in part by inhibition of a K+ current via postsynaptic 5-HT2A receptors. As mRNA for the two-pore-domain K+ channels TASK-1 (KCNK3) and TASK-3 (KCNK9) has been found in DVN, we investigated the possibility that 5-HT exerts its effects via inhibition of these K+ channels using whole-cell patch-clamp techniques. In current clamp, 5-HT (20 microM) elicited a depolarization by 5.1+/-1.5 mV and an increase in firing rate. In voltage clamp, 5-HT reduced the standing outward current (ISO) at -20 mV by 106+/-17 pA, inhibiting a conductance (reversal, -95+/-4 mV) which displayed Goldman-Hodgkin-Katz outward rectification, supportive of a TASK-like K+ current. Since TASK channels are modulated by extracellular pH (pHo), we next investigated the pH sensitivity of ISO in Hepes-buffered ACSF. At pHo 7.3, DVN exhibited an ISO of 147+/-15 pA at -20 mV. Acidification to pHo 6.3 reduced ISO to 85+/-13 pA, whereas raising pHo to 8.5 increased ISO to 216+/-26 pA. At pHo 7.3, ISO was inhibited by BaCl2 (IC50 465 microM), but unaffected by ZnCl2 (100 microM). 5-HT (10 microM) reduced ISO by 114+/-17 pA at pHo 7.3, but at pHo 6.3 the 5-HT-induced inhibition of ISO was significantly smaller. The present data suggest that the excitatory effects of 5-HT on DVN are mediated in part by inhibition of a TASK-like, pH-sensitive K+ conductance. The pharmacological profile of this conductance excludes TASK-3 homomers, but rather implicates TASK-1-containing channels.

Figure 1. Effects of 5-HT

A, whole-cell current-clamp recording of spontaneous activity of a dorsal vagal neurone (DVN). Bath-application of 5-HT (20 μm) for 2 min (filled bar) causes a reversible, small depolarization and concomitant increase in spontaneous action potential firing. B, in voltage clamp, 5-HT reduces the standing outward current (I_SO) at −20 mV command voltage. C, this effect is completely blocked by prior application of the 5-HT₂ receptor antagonist ketanserin (1 μm). Note the slow recovery of the 5-HT inhibition of I_SO after washout of ketanserin, and the rundown of I_SO over the course of the recording. D, mean effects of 5-HT and ketanserin in recordings similar to those shown in B and C; ‘wash’ indicates the current after removal of 5-HT immediately prior to application of ketanserin. The difference between the ‘control’ and ‘wash’ bar, which is not statistically significant, reflects the rundown of I_SO prior to ketanserin treatment. Number of cells (n) is given above the bars. **P < 0.01 versus control (unpaired t test). E, representative current–voltage relation (I–V) obtained from a DVN in the presence and absence of 5-HT (20 μm), and I–V for the 5-HT-inhibited current (I_control−I_5-HT). This current is well described by the Goldman-Hodgkin-Katz (GHK) current equation (dashed line).

Figure 2. pH dependence of I_SO

A, typical traces from a DVN recorded in voltage clamp in Hepes-buffered ACSF at different pH_o values. Acidification (pH_o 6.3) reduces I_SO at −20 mV, and alkalinization (pH_o 8.5) increases it. Depolarization from −120 to −20 mV from −120 mV activates a large slowly inactivating outward current. B, current amplitude at −20 mV measured over the course of a recording. C, mean I_SO at different pH values. Number of cells (n) is given above the bars. *P < 0.05; **P < 0.01 versus pH_o 7.3 (unpaired t test). D, representative I–V relations from a DVN obtained at different pH values. E, I–V of the acidosis-inhibited current is described by the GHK current equation (dashed line).

Figure 3. BaCl₂ sensitivity of the TASK-like current

A, the alkalinization-activated I_SO at −20 mV is inhibited by 1 mm BaCl₂. This effect is partially reversible. B, mean data from recordings as shown in A. Number of cells (n) are given above the bars. *P < 0.05 versus pH_o 8.5 (unpaired t test). C, concentration–response curve for BaCl₂ at pH_o 7.3. I/I_control is the current in the presence of BaCl₂ expressed as a fraction of the current prior to BaCl₂ application. The line is the best fit to the Hill equation using an IC₅₀ of 465 ± 11 μm and Hill coefficient of 2.01 ± 0.08.

Figure 4. BaCl₂ inhibition of both Kir and TASK-like currents

A, a low concentration (30 μm) of BaCl₂ inhibits primarily an inward K⁺ current in DVN. Increasing BaCl₂ to 1 mm inhibited mainly an outward current. B, mean current amplitude at a command potential of −20 and −120 mV in the absence and presence of 30 μm or 1 mm BaCl₂, respectively (all bars n = 5). *P < 0.05 versus 30 μm BaCl₂ at holding potential (V_H) −20 mV; *P < 0.05 versus control at V_H−120 mV (paired t test). C, lower panel, I–V relation of current inhibited by 30 μm BaCl₂, indicative of a strongly rectifying Kir channel; upper panel, I–V relation of current inhibited by the increase of BaCl₂ from 30 μm to 1 mm. This current is described well by the GHK current equation (continuous line).

Figure 5. Effects of ZnCl₂

A, I–V relations at pH_o 7.3 in the presence and absence of 100 μm ZnCl₂, and at pH_o 6.3. The TASK-3 blocker ZnCl₂ failed to inhibit the pH-sensitive K⁺ current. B, mean data from recordings as shown in A. Number of cells (n) is given above the bars.

Figure 6. Dependence of 5-HT effects on extracellular pH

A, modulation of I_SO at −20 mV in response to 5-HT at pH_o 6.3 and at pH_o 7.3. I_SO was measured every 30 s. B, mean inhibition of I_SO by 5-HT at the pH indicated. The number of cells (n) is given above the bars. **P < 0.01 versus control (pH_o 7.3) (paired t test).