Pflügers Archiv - European Journal of Physiology

, Volume 470, Issue 2, pp 327–337 | Cite as

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

  • Joohan Woo
  • Young Keul Jun
  • Yin-Hua Zhang
  • Joo Hyun Nam
  • Dong Hoon ShinEmail author
  • Sung Joon KimEmail author
Ion channels, receptors and transporters
Part of the following topical collections:
  1. Ion channels, receptors and transporters


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.


Two-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. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Bagriantsev SN, Peyronnet R, Clark KA, Honore E, Minor DL Jr (2011) Multiple modalities converge on a common gate to control K2P channel function. EMBO J 30:3594–3606. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Bagriantsev SN, Clark KA, Minor DL Jr (2012) Metabolic and thermal stimuli control K2P2.1 (TREK-1) through modular sensory and gating domains. EMBO J 31:3297–3308. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Beltran L, Beltran M, Aguado A, Gisselmann G, Hatt H (2013) 2-Aminoethoxydiphenyl borate activates the mechanically gated human KCNK channels KCNK 2 (TREK-1), KCNK 4 (TRAAK), and KCNK 10 (TREK-2). Front Pharmacol 4:63. PubMedPubMedCentralGoogle Scholar
  5. 5.
    Cabanos C, Wang M, Han X, Hansen SB (2017) A soluble fluorescent binding assay reveals PIP2 antagonism of TREK-1 channels. Cell Rep 20:1287–1294. CrossRefPubMedGoogle Scholar
  6. 6.
    Chemin J, Patel AJ, Duprat F, Lauritzen I, Lazdunski M, Honore E (2005) A phospholipid sensor controls mechanogating of the K+ channel TREK-1. EMBO J 24:44–53. CrossRefPubMedGoogle Scholar
  7. 7.
    Chemin J, Patel AJ, Duprat F, Sachs F, Lazdunski M, Honore E (2007) Up- and down-regulation of the mechano-gated K2P channel TREK-1 by PIP2 and other membrane phospholipids. Pflugers Arch 455:97–103. CrossRefPubMedGoogle Scholar
  8. 8.
    Enyedi P, Czirják G (2010) Molecular background of leak K+ currents: two-pore domain potassium channels. Physiol Rev 90:559–605. CrossRefPubMedGoogle Scholar
  9. 9.
    Gamper N, Rohacs T (2012) Phosphoinositide sensitivity of ion channels, a functional perspective. Subcell Biochem 59:289–333. CrossRefPubMedGoogle Scholar
  10. 10.
    Hille B, Dickson EJ, Kruse M, Vivas O, Suh BC (2015) Phosphoinositides regulate ion channels. Biochim Biophys Acta 1851:844–856. CrossRefPubMedGoogle Scholar
  11. 11.
    Honore E, Maingret F, Lazdunski M, Patel AJ (2002) An intracellular proton sensor commands lipid- and mechano-gating of the K+ channel TREK-1. EMBO J 21:2968–2976. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kim Y, Gnatenco C, Bang H, Kim D (2001) Localization of TREK-2 K+ channel domains that regulate channel kinetics and sensitivity to pressure, fatty acids and pHi. Pflugers Arch 442:952–960CrossRefPubMedGoogle Scholar
  13. 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. CrossRefPubMedGoogle Scholar
  14. 14.
    Logothetis DE, Petrou VI, Adney SK, Mahajan R (2010) Channelopathies linked to plasma membrane phosphoinositides. Pflugers Arch 460:321–341. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Lopes CM, Rohacs T, Czirjak G, Balla T, Enyedi P, Logothetis DE (2005) PIP2 hydrolysis underlies agonist-induced inhibition and regulates voltage gating of two-pore domain K+ channels. J Physiol 564:117–129. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Lopes CM, Remon JI, Matavel A, Sui JL, Keselman I, Medei E, Shen Y, Rosenhouse-Dantsker A, Rohacs T, Logothetis DE (2007) Protein kinase A modulates PLC-dependent regulation and PIP2-sensitivity of K+ channels. Channels (Austin) 1:124–134CrossRefGoogle Scholar
  17. 17.
    Maingret F, Lauritzen I, Patel AJ, Heurteaux C, Reyes R, Lesage F, Lazdunski M, Honore E (2000) TREK-1 is a heat-activated background K+ channel. EMBO J 19:2483–2491. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Mathie A, Veale EL (2015) Two-pore domain potassium channels: potential therapeutic targets for the treatment of pain. Pflugers Arch 467:931–943. CrossRefPubMedGoogle Scholar
  19. 19.
    McClenaghan C, Schewe M, Aryal P, Carpenter EP, Baukrowitz T, Tucker SJ (2016) Polymodal activation of the TREK-2 K2P channel produces structurally distinct open states. J Gen Physiol 147:497–505. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Murbartian J, Lei Q, Sando JJ, Bayliss DA (2005) Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels. J Biol Chem 280:30175–30184. CrossRefPubMedGoogle Scholar
  21. 21.
    Noel J, Sandoz G, Lesage F (2011) Molecular regulations governing TREK and TRAAK channel functions. Channels (Austin) 5:402–409. CrossRefGoogle Scholar
  22. 22.
    Park H, Kim EJ, Han J, Han J, Kang D (2016) Effects of analgesics and antidepressants on TREK-2 and TRESK currents. Korean J Physiol Pharmacol 20:379–385. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Patel AJ, Honore E, Maingret F, Lesage F, Fink M, Duprat F, Lazdunski M (1998) A mammalian two pore domain mechano-gated S-like K+ channel. EMBO J 17:4283–4290. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Patel AJ, Honore E, Lesage F, Fink M, Romey G, Lazdunski M (1999) Inhalational anesthetics activate two-pore-domain background K+ channels. Nat Neurosci 2:422–426. CrossRefPubMedGoogle Scholar
  25. 25.
    Patel AJ, Honore E (2001) Properties and modulation of mammalian 2P domain K+ channels. Trends Neurosci 24:339–346CrossRefPubMedGoogle Scholar
  26. 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. CrossRefPubMedGoogle Scholar
  27. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Renigunta V, Schlichthorl G, Daut J (2015) Much more than a leak: structure and function of K2p-channels. Pflugers Arch 467:867–894. CrossRefPubMedGoogle Scholar
  29. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 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. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Sandoz G, Bell SC, Isacoff EY (2011) Optical probing of a dynamic membrane interaction that regulates the TREK1 channel. Proc Natl Acad Sci U S A 108:2605–2610. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 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. CrossRefPubMedGoogle Scholar
  33. 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. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Department of Physiology, College of MedicineSeoul National UniversitySeoulKorea
  2. 2.Department of PhysiologyDongguk University College of MedicineGyeongjuRepublic of Korea
  3. 3.Department of PharmacologyYonsei UniversitySeoulKorea

Personalised recommendations