Identification of critical amino acids in the proximal C-terminal of TREK-2 K+ channel for activation by acidic pHi and ATP-dependent inhibition
- 377 Downloads
TWIK-related two-pore domain K+ channels (TREKs) are regulated by intracellular pH (pHi) and Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Previously, Glu306 in proximal C-terminal (pCt) of mouse TREK-1 was identified as the pHi-sensing residue. The direction of PI(4,5)P2 sensitivity is controversial, and we have recently shown that TREKs are inhibited by intracellular ATP via endogenous PI(4,5)P2 formation. Here we investigate the anionic and cationic residues of pCt for the pHi and ATP-sensitivity in human TREK-2 (hTREK-2). In inside-out patch clamp recordings (ITREK-2,i-o), acidic pHi-induced activation was absent in E332A and was partly attenuated in E335A. Neutralization of cationic Lys (K330A) also eliminated the acidic pHi sensitivity of ITREK-2,i-o. Unlike the inhibition of wild-type (WT) ITREK-2,i-o by intracellular ATP, neither E332A nor K330A was sensitive to ATP. Nevertheless, exogenous PI(4,5)P2 (10 μM) abolished ITREK-2 i-o in all the above mutants as well as in WT, indicating unspecific inhibition by exogenous PI(4,5)P2. In whole-cell recordings of TREK-2 (ITREK-2,w-c), K330A and E332A showed higher or fully active basal activity, showing attenuated or insignificant activation by 2-APB, arachidonic acid, or acidic pHe 6.9. ITREK-1,w-c of WT is largely suppressed by pHe 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 pHi sensitivity.
KeywordsTwo-pore K+ channel TREK-2 Intracellular pH ATP PI(4,5)P2
This work was supported by the National Research Foundation of Korea (NRF) grant (no. 2016R1D1A1A02937499) funded by the Korean Government (MOE) and by a grant from Seoul National University Hospital (2017).
- 1.Acosta C, Djouhri L, Watkins R, Berry C, Bromage K, Lawson SN (2014) TREK2 expressed selectively in IB4-binding C-fiber nociceptors hyperpolarize their membrane potentials and limits spontaneous pain. J Neurosci 34:1494–1509. https://doi.org/10.1523/JNEUROSCI.4528-13.2014 CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Lesage F, Terrenoire C, Romey G, Lazdunski M (2000) Human TREK2, a 2P domain mechano-sensitive K+ channel with multiple regulations by polyunsaturated fatty acids, lysophospholipids, and Gs, Gi, and Gq protein-coupled receptors. J Biol Chem 275:28398–28405. https://doi.org/10.1074/jbc.M002822200 CrossRefPubMedGoogle Scholar
- 26.Pereira V, Busserolles J, Christin M, Devilliers M, Poupon L, Legha W, Alloui A, Aissouni Y, Bourinet E, Lesage F, Eschalier A, Lazdunski M, Noel J (2014) Role of the TREK2 potassium channel in cold and warm thermosensation and in pain perception. Pain 155:2534–2544. https://doi.org/10.1016/j.pain.2014.09.013 CrossRefPubMedGoogle Scholar
- 27.Piechotta PL, Rapedius M, Stansfeld PJ, Bollepalli MK, Ehrlich G, Andres-Enguix I, Fritzenschaft H, Decher N, Sansom MS, Tucker SJ, Baukrowitz T (2011) The pore structure and gating mechanism of K2P channels. EMBO J 30:3607–3619. https://doi.org/10.1038/emboj.2011.268 CrossRefPubMedPubMedCentralGoogle Scholar
- 29.Sandoz G, Thummler S, Duprat F, Feliciangeli S, Vinh J, Escoubas P, Guy N, Lazdunski M, Lesage F (2006) AKAP150, a switch to convert mechano-, pH- and arachidonic acid-sensitive TREK K+ channels into open leak channels. EMBO J 25:5864–5872. https://doi.org/10.1038/sj.emboj.7601437 CrossRefPubMedPubMedCentralGoogle Scholar
- 30.Sandoz G, Douguet D, Chatelain F, Lazdunski M, Lesage F (2009) Extracellular acidification exerts opposite actions on TREK1 and TREK2 potassium channels via a single conserved histidine residue. Proc Natl Acad Sci U S A 106:14628–14633. https://doi.org/10.1073/pnas.0906267106 CrossRefPubMedPubMedCentralGoogle Scholar
- 32.Woo J, Shin DH, Kim HJ, Yoo HY, Zhang YH, Nam JH, Kim WK, Kim SJ (2016) Inhibition of TREK-2 K+ channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase. Pflugers Arch 468:1389–1402. https://doi.org/10.1007/s00424-016-1847-0 CrossRefPubMedGoogle Scholar
- 33.Zheng H, Nam JH, Pang B, Shin DH, Kim JS, Chun YS, Park JW, Bang H, Kim WK, Earm YE, Kim SJ (2009) Identification of the large-conductance background K+ channel in mouse B cells as TREK-2. Am J Physiol Cell Physiol 297:C188–C197. https://doi.org/10.1152/ajpcell.00052.2009 CrossRefPubMedGoogle Scholar