Inhibition of TREK-2 K(+) channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase.
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.
Department of Physiology, College of Medicine, Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, South Korea.
Joohan
J
Woo
Division of Natural Medical Sciences, College of Health Science, Chosun University, Gwang-Ju, 501-759, South Korea.
Dong Hoon
DH
Shin
Department of Physiology and Ion Channel Disease Research Center, College of Medicine, Dongguk University, Kyungju, 780-714, South Korea.
Hyun Jong
HJ
Kim
Chung-Ang University Red Cross College of Nursing, Seoul, 100-031, South Korea.
Hae Young
HY
Yoo
Department of Physiology, College of Medicine, Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, South Korea.
Yin-Hua
YH
Zhang
Department of Physiology and Ion Channel Disease Research Center, College of Medicine, Dongguk University, Kyungju, 780-714, South Korea.
Joo Hyun
JH
Nam
Department of Physiology and Ion Channel Disease Research Center, College of Medicine, Dongguk University, Kyungju, 780-714, South Korea.
Woo Kyung
WK
Kim
Department of Physiology, College of Medicine, Seoul National University, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, South Korea. physiolksj@gmail.com.
Sung Joon
SJ
Kim
D019870:1-Phosphatidylinositol 4-Kinase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology*; D015221:Potassium Channels / Q000378:metabolism*; D024683:Potassium Channels, Tandem Pore Domain / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*
D019269:Phosphatidylinositol 4,5-Diphosphate; D015221:Potassium Channels; D024683:Potassium Channels, Tandem Pore Domain; D047428:Protein Kinase Inhibitors; C103742:potassium channel protein TREK-1; D000255:Adenosine Triphosphate; D019870:1-Phosphatidylinositol 4-Kinase
ATP; PI kinase; Phosphatidylinositol 4,5-phosphate; TREK-2; Two-pore domain K+ channel