Dual regulatory effects of PI(4,5)P₂ on TREK-2 K⁺ channel through antagonizing interaction between the alkaline residues (K³³⁰ and R³⁵⁵⁻³⁵⁷) in the cytosolic C-terminal helix

TWIK-related two-pore domain K⁺ channel-2 (TREK-2) has voltageindependent activity and shows additional activation by acidic intracellular pH (pHi) via neutralizing the E³³² in the cytoplasmic C terminal (Ct). We reported opposite regulations of TREK-2 by phosphatidylinositol 4,5-bisphosphate (PIP₂) via the alkaline K³³⁰ and triple Arg residues (R³⁵⁵⁻³⁵⁷); inhibition and activation, respectively. The G³³⁴ between them appeared critical because its mutation (G³³⁴A) endowed hTREK-2 with tonic activity, similar to the mutation of the inhibitory K³³⁰ (K³³⁰A). To further elucidate the role of putative bent conformation at G³³⁴, we compared the dual mutation forms, K³³⁰A/G³³⁴A and G³³⁴A/³⁵⁵-⁷A, showing higher and lower basal activity, respectively. The results suggested that the tonic activity of G³³⁴A owes to a dominant influence from R³⁵⁵⁻⁷. Since there are additional triple Arg residues (³⁷⁷⁻⁹) distal to ³⁵⁵-⁷, we also examined the triple mutant (G³³⁴A/³⁵⁵-⁷A/³⁷⁷⁻⁹A) that showed tonic inhibition same with G³³⁴A/³⁵⁵-⁷A. Despite the state of tonic inhibition, the activation by acidic pHi was preserved in both G³³⁴A/³⁵⁵-⁷A and G³³⁴A/³⁵⁵-⁷A/³⁷⁷⁻⁹A, similar to the ³⁵⁵-⁷A. Also, the inhibitory effect of ATP could be commonly demonstrated under the activation by acidic pHi in ³⁵⁵-⁷A, G³³⁴A/³⁵⁵-⁷A, and G³³⁴A/³⁵⁵-⁷A/³⁷⁷⁻⁹A. These results suggest that the putative bent conformation at G³³⁴ is important to set the tug-of-war between K³³⁰ and ³⁵⁵-⁷ in the PIP₂-dependent regulation of TREK-2.