Cell experiment:

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].

ML335 is an activator of the two-pore domain potassium channels K2P2.1/TREK1 and K2P10.1/TREK2 (EC50s = 14.3 and 5.2 μM, respectively, in Xenopus oocytes).1 It is selective for K2P2.1/TREK1 and K2P10.1/TREK2 channels over K2P4.1/TRAAK channels. ML335 activates K2P2.1/TREK1 by binding to the C-type gate, which is the active site of TREK channels.

|1. Lolicato, M., Arrigoni, C., Mori, T., et al. K2P2.1(TREK-1): Activator complexes reveal a cryptic selectivity filter binding site. Nature 547(7663), 364-368 (2017).