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

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.

Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels

“…We used a voltage-sensitive phosphatase (VSP), which dephosphorylates PIP 2 into PIP. As previously described ( Woo et al, 2016 ), activation of this phosphatase induced first a weak increase of TREK1 current, then a sustained decrease, confirming the dual effect of PIP 2 on TREK1. Unlike TREK1, TRAAK is quickly inhibited by the depletion of PIP 2 , suggesting that TREK1, and TRAAK have different affinities for PIP 2 .…”

“…Data suggest that PIP 2 could be the negatively charged phospholipid of the plasma membrane involved in its binding to pCt. Exogenous application of PIP 2 activates TRAAK ( Lopes et al, 2005 ), inhibits TREK2 currents ( Zheng et al, 2009 ; Woo et al, 2016 ) and has a dual effect on TREK1 ( Chemin et al, 2007 ). However, a prolonged depletion of PIP 2 induced by a voltage-sensitive phosphatase leads to a decrease of TREK2 currents ( Woo et al, 2016 ).…”

“…Exogenous application of PIP 2 activates TRAAK ( Lopes et al, 2005 ), inhibits TREK2 currents ( Zheng et al, 2009 ; Woo et al, 2016 ) and has a dual effect on TREK1 ( Chemin et al, 2007 ). However, a prolonged depletion of PIP 2 induced by a voltage-sensitive phosphatase leads to a decrease of TREK2 currents ( Woo et al, 2016 ). Several positively charged residues (R297, K301, K302, K304, and R311) in the proximal C-ter of TREK1 seems crucial for PIP 2 interactions ( Chemin et al, 2005 ), a view also supported by the recent crystal structure of this channel ( Lolicato et al, 2017 ).…”

“…Finally, we tested the potential role of phosphatidylinositol-4, 5-bisphosphate (PIP 2 ) on channel activity. Previous studies have shown that this lipid, located in the inner leaflet of the plasma membrane, modulates TREK1 and TREK2 channels through an interaction with basic residues within pCt ( Chemin et al, 2005 ; Sandoz et al, 2011 ; Woo et al, 2016 , 2018 ; Lolicato et al, 2017 ). We used a voltage-sensitive phosphatase from Danio rerio (VSP), which dephosphorylates nearly all the PIP 2 in the membrane ( Okamura et al, 2009 ), to evaluate its role in the gating of TREK1 and TRAAK.…”

A regulatory domain in the K2P2.1 (TREK-1) carboxyl-terminal allows for channel activation by monoterpenes

“…A schematic model of the interplay between the different C-terminal domains and the sensitivity of K2P2.1 channels to various stimuli. According to the suggested models for K2P2.1 Honore, 2007) and K2P10.1 (Woo et al, 2018;Woo et al, 2016) channel regulation, the activity level of the channel is tightly related to the interaction of two intracellular C-terminal domains (a proximal one and a distal one) with the membrane. The proximal domain was proposed to contain a low-affinity site for PIP2 binding, while the distal domain was proposed to present a high-affinity site for PIP2 binding (Woo et al, 2018).…”