Identification of critical amino acids in the proximal C-terminal of TREK-2 K+ channel for activation by acidic pHi and ATP-dependent inhibition

TWIK-related two-pore domain K+ channels (TREKs) are regulated by intracellular pH (pH(i)) and Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2). Previously, Glu(306) in proximal C-terminal (pCt) of mouse TREK-1 was identified as the pH(i)-sensing residue. The direction of PI(4,5)P-2 sensitivity is controversial, and we have recently shown that TREKs are inhibited by intracellular ATP via endogenous PI(4,5)P-2 formation. Here we investigate the anionic and cationic residues of pCt for the pH(i) and ATP-sensitivity in human TREK-2 (hTREK-2). In inside-out patch clamp recordings (I-TREK-2,I-i-o), acidic pH(i)-induced activation was absent in E332A and was partly attenuated in E335A. Neutralization of cationic Lys (K330A) also eliminated the acidic pH(i) sensitivity of I-TREK-2,I-i-o. Unlike the inhibition of wild-type (WT) I-TREK-2,I-i-o by intracellular ATP, neither E332A nor K330A was sensitive to ATP. Nevertheless, exogenous PI(4,5)P-2 (10 mu M) abolished ITREK-2 i-o in all the above mutants as well as in WT, indicating unspecific inhibition by exogenous PI(4,5)P-2. In whole-cell recordings of TREK-2 (I-TREK-2,I-w-c), K330A and E332A showed higher or fully active basal activity, showing attenuated or insignificant activation by 2-APB, arachidonic acid, or acidic pH(e) 6.9. I-TREK-1,I-w-c of WT is largely suppressed by pH(e) 6.9, and the inhibition is slightly attenuated in K312A and E315A. The results show concerted roles of the oppositely charged Lys and Glu in pCt for the ATP-dependent low basal activity and pH(i) sensitivity.