Activation of TREK currents by riluzole in three subgroups of cultured mouse nodose ganglion neurons

Abstract

Two-pore domain potassium channels (K2P) constitute major candidates for the regulation of background potassium currents in mammalian cells. Channels of the TREK subfamily are also well positioned to play an important role in sensory transduction due to their sensitivity to a large number of physiological and physical stimuli (pH, mechanical, temperature). Following our previous report describing the molecular expression of different K2P channels in the vagal sensory system, here we confirm that TREK channels are functionally expressed in neurons from the mouse nodose ganglion (mNG). Neurons were subdivided into three groups (A, Ah and C) based on their response to tetrodotoxin and capsaicin. Application of the TREK subfamily activator riluzole to isolated mNG neurons evoked a concentration-dependent outward current in the majority of cells from all the three subtypes studied. Riluzole increased membrane conductance and hyperpolarized the membrane potential by approximately 10 mV when applied to resting neurons. The resting potential was similar in all three groups, but C cells were clearly less excitable and showed smaller hyperpolarization-activated currents at -100 mV and smaller sustained currents at -30 mV. Our results indicate that the TREK subfamily of K2P channels might play an important role in the maintenance of the resting membrane potential in sensory neurons of the autonomic nervous system, suggesting its participation in the modulation of vagal reflexes.

Fig 1. Basic properties of mNG neurons in culture.

(A) Different micrographs (after 24 h. in culture) were taken with an inverted phase contrast microscope. The brightness of the membrane contour serves as indication of cell viability to perform patch-clamp experiments. (B) Frequency distribution of capacitance values for the experimental sample. Shapiro-Wilk test fails to detect a normal distribution (P<0.001, n = 81). (C) Frequency distribution of resting membrane potential values for the experimental sample. Shapiro-Wilk test reports a Gaussian distribution (P = 0.57, n = 81).

Fig 2. Riluzole-activated outward current and hyperpolarization.

(A) Short (50 ms) voltage steps (to -45 mV) were applied at high frequency (50 Hz) to detect changes of membrane conductance in the presence of 300 μM riluzole and cocktail (holding potential -30 mV). (B) Dose-response curve representing the current activated by riluzole with the membrane clamped at -30 mV, in the presence of the cocktail. Estimated EC₅₀ was 392.2±182.8 μM. Number of cells for each concentration are specified under the points. (C) Hyperpolarization induced by the application of riluzole, for 3 minutes, on the resting membrane potential (V_m) in bridge mode-like experiments (I = 0). Note that this recording comes from a different cell than that depicted in A. (D) Current–voltage relationships for riluzole-induced currents in the presence of cocktail, obtained in response to negatively progressing voltage ramps. The current obtained in the control was subtracted from that obtained in the presence of 300 μM riluzole. Note that the strong outward rectification obtained in standard solutions (E_K = -90.7 mV) disappeared when symmetrical concentrations of potassium were used (E_K = 0 mV). Recordings were acquired from two different cells.

Fig 3. Riluzole-activated outward current is mediated by TREK channels.

(A) Short (50 ms) voltage steps (to -45 mV) were applied at high frequency (50 Hz) to detect changes of membrane conductance in the presence of 30 μM ML67-33 and the cocktail solution (holding potential -30 mV). Note the clear inhibition of the I_ML by fluoxetine. (B) Short (50 ms) voltage steps (to -45 mV) were applied at high frequency (50 Hz) to detect changes of membrane conductance in the presence of 3 μM BL-1249 and cocktail B (holding potential -30 mV). Note the clear inhibition of the I_BL by fluoxetine. (C) Short (50 ms) voltage steps (to -45 mV) were applied at high frequency (50 Hz) to detect changes of membrane conductance in the presence of 300 μM Riluzole and cocktail B (holding potential -30 mV). Note the clear inhibition of the I_Ril by fluoxetine. (D) After 1.5 hour of incubation in 1 μM spadin, short (50 ms) voltage steps (to -45 mV) were applied at high frequency (50 Hz) to detect changes of membrane conductance in the presence of 300 μM riluzole and cocktail B (holding potential -30 mV). Note that the decrease of I_Ril in comparison to control conditions (C). (E) Summary bars describing the inhibitory effect that 1.5-hour incubation of the TREK-1 inhibitor spadin exert on the current-activated by riluzole.

Fig 4. Riluzole (100 μM) is a potent inhibitor of human TRESK channels.

(A) Bath application of riluzole (100 μM, black bar) rapidly and reversible inhibited TRESK current measured at -40 mV. (B) Representative traces of TRESK currents in a single cell evoked using the “step ramp” protocol described in the methods in the absence (black) and presence (red) of riluzole (100 μM). (C) Representative current-voltage relationships for TRESK channels in a single cell in the absence (black) and presence (red) of riluzole (100 μM). (D) Inhibition of TRESK channel current at -40 mV by riluzole (100 μM) in 8 individual cells from 3 different recording days (gray dots). Error bars represent the mean ± SEM.

Fig 5. Effect of riluzole based on sensitivity to capsaicin.

(A) Neuron unresponsive to capsaicin: (A₁) Response to 1 μM capsaicin (V_m = -60 mV). (A₂) Response of the same cell to 300 μM riluzole in the presence of the cocktail (V_m = -30 mV). (B) Neuron responsive to capsaicin: (B₁) Response to 1 μM capsaicin (V_m = -60 mV). (B₂) Response of the same cell to 300 μM riluzole in the presence of the cocktail (V_m = -30 mV). (C) Resting membrane potential levels are not dependent on capsaicin sensitivity (left). The steady-state outward current with the membrane clamped at -30 mV (I_-30) is significantly higher in capsaicin-insensitive neurons (***P<0.001, right). (D) The number of action potentials in response to increasing one second current injections is smaller in capsaicin-insensitive neurons (*P<0.05).

Fig 6. Effect of riluzole based on sensitivity to TTX.

(A) Neuron with only TTX-sensitive (TTX-S) currents: (A₁) Voltage-dependent inward currents in the presence of TEA, 4-AP, CsCl, and CdCl₂, evoked with a voltage step from -80 to 0 mV (300 ms), before and after the addition of 0.5 μM TTX. (A₂) Response of the same cell to 300 μM riluzole in the presence of the cocktail (V_m = -30 mV). (B) Neuron with both TTX-S and TTX-resistant currents (TTX-SR): (B₁) Voltage-dependent inward currents in the presence of TEA, 4-AP, CsCl, and CdCl₂, evoked with a voltage step from -80 to 0 mV (300 ms) before and after the addition of 0.5 μM TTX). (B₂) Response of the same cell to 300 μM riluzole in the presence of the cocktail (V_m = -30 mV). (C) Resting membrane potential levels are not dependent on TTX sensitivity (left). Constant outward current with the membrane clamped at -30 mV is significantly higher in TTX-S neurons (*P<0.05, right). (D) Action potential firing in response to increasing one-second current injections is not dependent on TTX sensitivity.

Fig 7. Characterization of three subpopulations of neurons in the mNG and response to riluzole.

A-type (A), Ah-type (B) and C-type (C) neurons are represented. (1) Only C-type mNG neurons respond to the application of 1 μM capsaicin (holding potential -60 mV). (2) Only A-type neurons show no TTX-resistant inward currents after a voltage step from -80 to 0 mV. (3) There are no significant differences in the outward current evoked by 300 μM riluzole among the different groups of neurons (holding potential -30 mV, in the presence of the cocktail). (4) Voltage ramps from -30 to -100 mV (10 mV/sec) were applied to study the magnitude of the h-current, revealed at hyperpolarized potentials after the application of 1 mM CsCl. All recordings in voltage-clamp experiments for each cell type are from the same neuron. (5) Changes in membrane potential after the injection of different current intensities (-100, 0, 100 and 200 (only C-type) pA) in the different groups of cells. Note the larger sag (arrow) seen after the hyperpolarizing pulse in A- and Ah-type cells, compared to C-type. Note that C-type neurons do not fire with current intensities under 200 pA. (6) Enlarged action potentials (first action potential in the depolarizing pulse in (5)) are represented to highlight their different duration among groups. Note that the measurement of the action potential duration is performed at half amplitude, where the characteristic hump of C-type cells is clearly distinguishable.