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Inhibition of TREK-2 K+ channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase

  • Ion channels, receptors and transporters
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Abstract

TWIK-related two-pore domain K+ channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P2 and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P2 on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (ITREK-1,w-c and ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (ITREK-1,i-o and ITREK-2,i-o). Steady state ITREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P2 inhibited the spontaneously increased ITREKs,i-o, suggesting that intrinsic PI(4,5)P2 maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited ITREK-2,i-o. Application of PI(4,5)P2 after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of ITREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P2 phosphatase, sustained depolarization increased ITREK-2,w-c initially (<5 s) but then decreased the current below the control level. In HEK293T cells coexpressing TREK-2 and type 3 muscarinic receptor, application of carbachol induced transient activation and sustained suppression of ITREK-2,w-c and cell-attached ITREK-2. The inhibition of TREK-2 by unspecific electrostatic quenching, extensive dephosphorylation, or sustained hydrolysis of PI(4,5)P2 suggests the existence of dual regulatory modes that depend on PI(4,5)P2 concentration.

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Acknowledgments

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP; Nos. 2007–0056092 and 2011–0017370) and by the Education and Research Encouragement Fund from Seoul National University Hospital (2016).

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Authors and Affiliations

  1. Department of Physiology, College of Medicine, Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, South Korea

    Joohan Woo, Yin-Hua Zhang & Sung Joon Kim

  2. Division of Natural Medical Sciences, College of Health Science, Chosun University, Gwang-Ju, 501-759, South Korea

    Dong Hoon Shin

  3. Department of Physiology and Ion Channel Disease Research Center, College of Medicine, Dongguk University, Kyungju, 780-714, South Korea

    Hyun Jong Kim, Joo Hyun Nam & Woo Kyung Kim

  4. Chung-Ang University Red Cross College of Nursing, Seoul, 100-031, South Korea

    Hae Young Yoo

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  1. Joohan Woo
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Correspondence to Sung Joon Kim.

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Woo, J., Shin, D.H., Kim, H.J. et al. Inhibition of TREK-2 K+ channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase. Pflugers Arch - Eur J Physiol 468, 1389–1402 (2016). https://doi.org/10.1007/s00424-016-1847-0

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  • DOI: https://doi.org/10.1007/s00424-016-1847-0

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