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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)P and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P 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 and ) or by membrane excision for inside-out patch clamping without ATP (I and I). Steady state I 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)P inhibited the spontaneously increased I, suggesting that intrinsic PI(4,5)P 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. Application of PI(4,5)P after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of I at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P phosphatase, sustained depolarization increased I 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 and cell-attached I. The inhibition of TREK-2 by unspecific electrostatic quenching, extensive dephosphorylation, or sustained hydrolysis of PI(4,5)P suggests the existence of dual regulatory modes that depend on PI(4,5)P concentration. [ABSTRACT FROM AUTHOR]

Two-pore domain K + (K 2P ) channels are thought to be druggable targets. However, only a few agents specific for K 2P channels have been identified, presumably due to the lack of an efficient screening system. To develop a new high-throughput screening (HTS) system targeting these channels, we have established a HEK293-based "test cell" expressing a mutated Na + channel (Nav1.5) with markedly slowed inactivation, as well as a K + channel (Kir2.1) that sets the membrane potential quite negative, close to K + equilibrium potential. We found in this system that Kir2.1 block by 100 μM Ba 2+ application consistently elicited a large depolarization like a long-lasting action potential. This maneuver resulted in cell death, presumably due to the sustained Na + influx. When either the TWIK-related acid-sensitive K + (TASK)-1 or TASK-3 channel was expressed in the test cells, Ba 2+ -induced cell death was markedly weakened. Stronger activation of TASK-1 by extracellular acidification further decreased the cell death. In contrast, the presence of K 2P channel blockers enhanced cell death. IC 50 values for TASK-1 and/or TASK-3 blockers acquired by measurements of relative cell viability were comparable to those obtained using patch-clamp recordings. Both blockers and openers of K 2P channels can be accurately assessed with high efficiency and throughput by this novel HTS system.

TWIK-related two-pore domain K + channels (TREKs) are activated by acidic intracellular pH (pH i ), membrane stretch, temperature, and arachidonic acid (AA). Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) exerts concentration-dependent biphasic regulations, which have been observed: inhibition by high PIP 2 , activation by partial decrease of PIP 2 , and inhibition by depletion of PIP 2 . Consistently, the stimulation of voltage-sensitive PIP 2 phosphatase (Dr-VSP) induces initial activation and subsequent inhibition of TREKs. Lys in the proximal C-terminus (pCt) is responsible for the inhibition by high PIP 2 , which is generated by phosphatidylinositol kinases with ATP; its neutralizing mutation [K 330 A of human TREK-2 (hTREK-2)] induces tonic high activity, irrespective of ATP. Here we focus on triple successive Arg in pCt (R3-pCt) as a candidate region for the stimulatory regulation by lower PIP 2 . Their neutralized mutant (R3A-pCt; RRR 340-2 A and RRR 355-7 A in hTREK-1 and -2, respectively) showed negligible basal current and was not affected by ATP removal or by Dr-VSP activation. Phosphatidic acid, a phospholipid agonist of TREKs, did not activate R3A-pCt. In contrast, acidic pH i , AA, and high temperature activated R3A-pCt normally, whereas activation by membrane stretch was attenuated. In hTREK-2, combined neutralizations of the inhibitory K 330 and R3-pCt (K 330 A/RRR 355-7 A) did not recover the suppressed current. In contrast, combined neutralization of pH i -sensing Glu (E 332 A/R 355-7 A) induced tonic high current and no further activation by pH i . Interestingly, when the Gly between K 330 /E 332 and R3-pCt was mutated (G 334 A), hTREK-2 was tonic activated with reversed responses to ATP and acidic pH i . Therefore, we propose that the PIP 2 -dependent converse regulation of TREKs by Lys and R3-pCt with Gly implies structural flexibility.