Triple arginine residues in the proximal C-terminus of TREK K+ channels are critical for biphasic regulation by phosphatidylinositol 4,5-bisphosphate

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.