Enhancement of Current through Trek1 Two Pore Domain Channels by Flufenamic Acid

Abstract

Two pore domain potassium (K2P) channels are responsible for background currents that regulate neuronal membrane potential and excitability. It has been shown that TREK1 K2P channels are implicated in pain and can be activated by flufenamic acid (FFA). The aim of this study was to investigate the mechanism of FFA on TREK1 channels.tsA201 cells were transiently transfected with wild type and mutated TREK1 channels. The whole cell patch clamp technique was used to obtain current recordings. Homology models for TREK1 channels were constructed using Modeller 9v8.FFA (100μM) activated TREK1 channels by 207 ± 50% (n=7). From the model of TREK1, it was predicted that the inner helix residues (A286 and G171) face the ion conductance pathway of TREK1 pore near its intracellular terminus. Mutating A286 and G171 to a bulky F residue, significantly reduced the TREK1 current (WT: 533 ± 87pA, (n=7); A286F: 54 ± 5pA (n=8); G171F: 112 ± 19pA (n=17)). However, FFA (100μM) recovered these reduced currents. TREK1 A286F current was enhanced by 823 ± 275% (n=9); and TREK1 G171F by 523 ± 181% (n=7). Consequently, it was hypothesised that FFA induces a counter-clockwise helical rotation that removes the bulky F side chain from the ion pathway. Accordingly, A287 may move to occupy the A286 pore position. Consistent with this hypothesis, the A287F mutation abolished the FFA effect on TREK1 (enhancement: 7 ± 8%, n=8). Moreover, although, increasing pHext to 8.4 enhanced TREK1 current by 49 ± 6% (n=7), it did not substantially recover the reduced TREK1 A286F current (enhancement: 87 ± 13%, n=6).These results indicate that FFA may induce conformational changes at the TREK1 inner helix consistent with a counter clockwise helical rotation, which may contribute to TREK1 gating.