ML335 is a selective activator of TREK-1 and TREK-2.

Xenopus oocyte two-electrode voltage-clamp measurements show that ML335 and ML402 activate K2P2.1 and K2P10.1 but not K2P4.1 (14.3±2.7?μM, K2P2.1-ML335; 13.7±7.0?μM, K2P2.1-ML402; 5.2±0.5?μM, K2P10.1-ML335; and 5.9±1.6?μM, K2P10.1-ML402). Swapping the Lys271 equivalent between K2P2.1 and K2P4.1 results in a clear phenotype reversal for ML335 and M402 activation. ML335 and ML402 activate K2P2.1 in HEK293 cells similar to their effects in Xenopus oocytes (5.2±0.8?μM and 5.9±1.6?μM for ML335 and ML402, respectively (n≥3))

Mouse K2P2.1, human K2P4.1, and mutants are expressed from a previously described pIRES2-EGFP vector in HEK293T cells (ATTC). 70% confluent cells are transfected (in 35-mm diameter wells) with LipofectAMINE 2000 for 6?h, and plated onto coverslips coated with Matrigel. Effects of ML335, ML402 and arachidonic acid on K2P2.1 current at 0?mV are measured by whole-cell patch-clamp experiments 24?h after transfection. Acquisition and analysis are performed using pCLAMP9 and an Axopatch 200B amplifier. Pipette resistance ranges from 1 to 1.5?MΩ. Pipette solution contains the following: 145?mM KCl, 3?mM MgCl2, 5?mM EGTA and 20?mM HEPES (pH 7.2 with KOH). Bath solution contains the following: 145?mM NaCl, 5?mM KCl, 1?mM CaCl2, 3?mM MgCl2 and 20?mM HEPES (pH 7.4 with NaOH). K2P2.1 currents are elicited by a 1?s ramp from -100 to +50?mV from a -80?mV holding potential. After stabilization of the basal current, ML335 and ML402 are perfused at 200?mL per hour until potentiation is stably reached[1].