Inhibition of TREK-2 K⁺ channels by PI(4,5)P₂: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase

TWIK-related two-pore domain K⁺ channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P₂ and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P₂ on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (I_TREK-1,w-c and _ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (I_TREK-1,i-o and I_TREK-2,i-o). Steady state I_TREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P₂ inhibited the spontaneously increased I_TREKs,i-o, suggesting that intrinsic PI(4,5)P₂ maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited I_TREK-2,i-o. Application of PI(4,5)P₂ after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of I_TREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P₂ phosphatase, sustained depolarization increased I_TREK-2,w-c initially (<5 s) but then decreased the current below the control level. In HEK293T cells coexpressing TREK-2 and type 3 muscarinic receptor, application of carbachol induced transient activation and sustained suppression of I_TREK-2,w-c and cell-attached I_TREK-2. The inhibition of TREK-2 by unspecific electrostatic quenching, extensive dephosphorylation, or sustained hydrolysis of PI(4,5)P₂ suggests the existence of dual regulatory modes that depend on PI(4,5)P₂ concentration.