Differential sensitivity of TREK-1, TREK-2 and TRAAK background potassium channels to the polycationic dye ruthenium red

Abstract

BACKGROUND AND PURPOSE: Pharmacological separation of the background potassium currents of closely related K2P channels is a challenging problem. We previously demonstrated that ruthenium red (RR) inhibits TASK-3 (K2 P 9.1), but not TASK-1 (K2 P 3.1) channels. RR has been extensively used to distinguish between TASK currents in native cells. In the present study, we systematically investigate the RR sensitivity of a more comprehensive set of K2 P channels. EXPERIMENTAL APPROACH: K+ currents were measured by two-electrode voltage clamp in Xenopus oocytes and by whole-cell patch clamp in mouse dorsal root ganglion (DRG) neurons. KEY RESULTS: RR differentiates between two closely related members of the TREK subfamily. TREK-2 (K2 P 10.1) proved to be highly sensitive to RR (IC50 = 0.2 muM), whereas TREK-1 (K2 P 2.1) was not affected by the compound. We identified aspartate 135 (D135) as the target of the inhibitor in mouse TREK-2c. D135 lines the wall of the extracellular ion pathway (EIP), a tunnel structure through the extracellular cap characteristic for K2 P channels. TREK-1 contains isoleucine in the corresponding position. The mutation of this isoleucine (I110D) rendered TREK-1 sensitive to RR. The third member of the TREK subfamily, TRAAK (K2 P 4.1) was more potently inhibited by ruthenium violet, a contaminant in some RR preparations, than by RR. DRG neurons predominantly express TREK-2 and RR-resistant TREK-1 and TRESK (K2 P 18.1) background K+ channels. We detected the RR-sensitive leak K+ current component in DRG neurons. CONCLUSIONS AND IMPLICATIONS: We propose that RR may be useful for distinguishing TREK-2 (K2P 10.1) from TREK-1 (K2P 2.1) and other RR-resistant K2 P channels in native cells.