Dual regulatory effects of PI(4,5)P(2) on TREK-2 K(+) channel through antagonizing interaction between the alkaline residues (K(330) and R(355-357)) in the cytosolic C-terminal helix

TWIK-related two-pore domain K(+) channel-2 (TREK-2) has voltage-independent activity and shows additional activation by acidic intracellular pH (pH(i)) via neutralizing the E(332) in the cytoplasmic C terminal (Ct). We reported opposite regulations of TREK-2 by phosphatidylinositol 4,5-bisphosphate (PIP(2)) via the alkaline K(330) and triple Arg residues (R(355-357)); inhibition and activation, respectively. The G(334) between them appeared critical because its mutation (G(334)A) endowed hTREK-2 with tonic activity, similar to the mutation of the inhibitory K(330) (K(330)A). To further elucidate the role of putative bent conformation at G(334), we compared the dual mutation forms, K(330)A/G(334)A and G(334)A/R(355-7)A, showing higher and lower basal activity, respectively. The results suggested that the tonic activity of G(334)A owes to a dominant influence from R(355-7). Since there are additional triple Arg residues (R(377-9)) distal to R(355-7), we also examined the triple mutant (G(334)A/R(355-7)A/R(377-9)A) that showed tonic inhibition same with G(334)A/R(355-7)A. Despite the state of tonic inhibition, the activation by acidic pH(i) was preserved in both G(334)A/R(355-7)A and G(334)A/R(355-7)A/R(377-9)A, similar to the R(355-7)A. Also, the inhibitory effect of ATP could be commonly demonstrated under the activation by acidic pH(i) in R(355-7)A, G(334)A/R(355-7)A, and G(334)A/R(355-7)A/R(377-9)A. These results suggest that the putative bent conformation at G(334) is important to set the tug-of-war between K(330) and R(355-7) in the PIP(2)-dependent regulation of TREK-2.