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].

参考文献:

[1]. Lolicato M, et al. K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site. Nature. 2017 Jul 20;547(7663):364-368.