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 (PIP₂) exerts concentration-dependent biphasic regulations, which have been observed: inhibition by high PIP₂, activation by partial decrease of PIP₂, and inhibition by depletion of PIP₂. Consistently, the stimulation of voltage-sensitive PIP₂ 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 PIP₂, which is generated by phosphatidylinositol kinases with ATP; its neutralizing mutation [K³³⁰A 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 PIP₂. Their neutralized mutant (R3A-pCt; RRR^340-2A and RRR^355-7A 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 K³³⁰ and R3-pCt (K³³⁰A/RRR^355-7A) did not recover the suppressed current. In contrast, combined neutralization of pH_i-sensing Glu (E³³²A/R^355-7A) induced tonic high current and no further activation by pH_i. Interestingly, when the Gly between K³³⁰/E³³² and R3-pCt was mutated (G³³⁴A), hTREK-2 was tonic activated with reversed responses to ATP and acidic pH_i. Therefore, we propose that the PIP₂-dependent converse regulation of TREKs by Lys and R3-pCt with Gly implies structural flexibility.

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