The Neuroprotective Agent Riluzole Activates the Two P Domain K+ Channels TREK-1 and TRAAK

Abstract

Riluzole (RP 54274) is a potent neuroprotective agent with anticonvulsant, sedative, and anti-ischemic properties. It is currently used in the treatment of amyotrophic lateral sclerosis. This article reports that riluzole is an activator of TREK-1 and TRAAK, two important members of a new structural family of mammalian background K channels with four transmembrane domains and two pore regions. Whereas riluzole activation of TRAAK is sustained, activation of TREK-1 is transient and is followed by an inhibition. The inhibitory process is attributable to an increase of the intracellular cAMP concentration by riluzole that produces a protein kinase A-dependent inhibition of TREK-1. Mutants of TREK-1 lacking the Ser residue where the kinase A phosphorylation takes place are activated in a sustained manner by riluzole. TRAAK is permanently activated by riluzole because, unlike TREK-1, it lacks the negative regulation by cAMP. K channels are widely distributed membrane proteins that are involved in diverse cellular functions (Hille, 1992) and present a large variety of properties (conductance, activation and inactivation kinetics, regulation, pharmacology and molecular structure). A new structural family of K channels has been cloned recently from mammals (Lesage and Lazdunski, 1999). These channels contain four transmembrane (TM) domains and two P domains in the same protein subunit instead of one for the voltage-gated and inward-rectifier K channels. The properties, as well as the wide tissue distribution of these two P domain K channels, suggest that they are background channels involved in the generation and modulation of the resting membrane potential in various cell types (Fink et al., 1996, 1998; Lesage et al., 1996; Duprat et al., 1997; Reyes et al., 1998; Lesage and Lazdunski, 1999). A particularly interesting subclass of two P domain K channels is represented by TREK-1 and TRAAK, which are K channels that are activated both by arachidonic acid and by membrane stretch (Patel et al., 1998; Maingret et al., 1999). In addition, TREK-1 activity is also modulated by cAMP (inhibition) and by variations of internal pH (activation at acidic PHi) (Patel et al., 1998; Maingret et al., 1999). Riluzole (RP 54274) is a neuroprotective agent with anticonvulsant, sedative and anti-ischemic properties (Malgouris et al., 1989; Romettino et al., 1991; Stutzmann et al., 1991; Pratt et al., 1992; Bryson et al., 1996). It is particularly effective in preventing spinal cord injury, a devastating complication of thoracoabdominal aortic surgery (Lang-Lazdunski et al., 1999). It is also very effective in retinal ischemia in models of glaucoma (Ettaiche et al., 1999). It is currently used in the treatment of amyotrophic lateral sclerosis (Bensimon et al., 1994). It has anesthetic properties at high concentrations (Mantz et al., 1992; Maclver et al., 1996). An important part of the mechanism of riluzole is probably linked to the fact that it inhibits synaptic glutamate release. This inhibition could be partly attributable to the riluzoleinduced inhibition of voltage-dependent sodium channels (Doble, 1996). However, another mechanism of inhibition of glutamate release is the possible activation of background K channels (Fink et al., 1998), which would lead to robust polarization, possibly both at presynaptic and at postsynaptic levels. This article reports that riluzole activates TREK-1 and TRAAK. It analyzes the mechanistic reasons that result in a sustained activation of TRAAK by riluzole whereas TREK-1 is first transiently activated and then inhibited. Materials and Methods Molecular Biology. Reports on the cloning of TREK-1 and TRAAK and the construction of pIRES-CD8-TREK-1 and pIRESCD8-TRAAK have been published previously (Fink et al., 1996, 1998). We have also previously reported the construction of the protein kinase A (PKA) mutants (Patel et al., 1998). Patch-Clamp Recordings in Transfected COS Cells. COS cells were seeded at a density of 20,000 cells per 35-mm dish 24 h 1 This work was supported by the Centre National de la Recherche Scientifique and the Association Française Contre les Myopathies. ABBREVIATIONS: PDE, phosphodiesterase; PKA, protein kinase A; 8CPT, 8-(4-chlorophenylthio); IBMX, isobutyl methyl xanthine; DNP, dinitrophenol; MCD, mast cell degranulating. 0026-895X/00/050906-07$3.00/0 MOLECULAR PHARMACOLOGY Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics MOL 57:906–912, 2000 /13030/816881 906 at A PE T Jornals on M ay 1, 2017 m oharm .aspeurnals.org D ow nladed from before transfection. Cells were then transiently transfected by the classic DEAE-dextran method with 1 mg of TREK-1, TREK-1 C1, and TRAAK or 0.1 mg of PKA I and PKA II plasmids per 35-mm dish. Transiently transfected cells were visualized 48 h after transfection using the anti-CD8 antibody coated beads method. For whole-cell and excised-patch recordings, the internal solution contained 150 mM KCl, 3 mM MgCl2, 5 mM EGTA, and 10 mM HEPES at pH 7.2 with KOH, and the external solution 150 mM NaCl, 5 mM KCl, 3 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES at pH 7.4 with NaOH. Cells were superfused continuously with a microperfusion system during the experiments (0.1 ml/min) performed at room temperature. A RK400 patch-clamp amplifier (Bio-Logic, Grenoble, France) was used for whole-cell as well as single-channel recordings. Data were analyzed using pClamp software (Axon Instruments, Foster City, CA). Stock solutions of riluzole (0.1 M) were made in dimethylsulfoxide; the final solutions were made regularly before use. The variability of the results are expressed as the standard error of the mean (S.E.M.) with n indicating the number of cells tested contributing to the mean. Statistical significance was assayed using Student’s t test with a confidence level of 0.05, 0.01, or 0.001 indicated with *, **, or ***, respectively. cAMP Radioimmunoassay. COS cells were maintained in DMEM (Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum. Twenty-four to 48 h before the assay, cells were dissociated and 2.10 cells were plated per 35-mm dish. When the half-confluency was reached, the cells were briefly washed, then preincubated in a solution without drugs. After 10 min, this solution was removed and replaced by an equivalent solution containing the drug. For the washing, preincubation and incubation steps, the solution used was the external medium for electrophysiology. cAMP was extracted by adding 1 ml of 65% ethanol (220°C) on the cells immediately after removal of the incubation solution. The plates were placed at 220°C. After 3 h, the ethanol solution was collected and briefly centrifuged to eliminate cell fragments, then speed-vacuumed dried. Pellets were reconstituted with 200 ml of assay solution, and the quantification of cAMP was performed by using a commercial radioimmunoassay kit (Immunotech, Luminy, France). Phosphodiesterase (PDE) Assays. The PDE assays were performed using the procedures described by Nicholson et al. (1989) for bovine brain PDE I, Torphy et al. (1992) for human differentiated U-937 cell PDE II, Weishaar et al. (1986) for human platelet PDE III, and Torphy et al. (1992) for human U-937 cell PDE IV. The reactions were conducted at 30°C for 30 min using [H]cAMP as substrate (0.1 mM for PDE III and 1 mM for PDE I, II, and IV). 59-[H]AMP was recovered, then the radioactivity was measured by scintillation. For each assay, the effect of riluzole was tested at 100 mM in duplicate. In each experiment, an inhibitory compound of reference was tested simultaneously as control (8-methoxy-IBMX for PDEI, erythro-9–3(2-hydroxynonyl) adenine for PDEII, milrinone for PDE III, and rolipram for PDE IV).