Optical probing of a dynamic membrane interaction that regulates the TREK1 channel

Abstract

TREK channels produce background currents that regulate cell excitability. These channels are sensitive to a wide variety of stimuli including polyunsaturated fatty acids (PUFAs), phospholipids, mechanical stretch, and intracellular acidification. They are inhibited by neurotransmitters, hormones, and pharmacological agents such as the antidepressant fluoxetine. TREK1 knockout mice have impaired PUFA-mediated neuroprotection to ischemia, reduced sensitivity to volatile anesthetics, altered perception of pain, and a depression-resistant phenotype. Here, we investigate TREK1 regulation by Gq-coupled receptors (GqPCR) and phospholipids. Several reports indicate that the C-terminal domain of TREK1 is a key regulatory domain. We developed a fluorescent-based technique that monitors the plasma membrane association of the C terminus of TREK1 in real time. Our fluorescence and functional experiments link the modulation of TREK1 channel function by internal pH, phospholipid, and GqPCRs to TREK1-C-terminal domain association to the plasma membrane, where increased association results in greater activity. In keeping with this relation, inhibition of TREK1 current by fluoxetine is found to be accompanied by dissociation of the C-terminal domain from the membrane.T REK1 is a two-pore-domain K + (K 2P ) channel that produces a nearly time-and voltage-independent background current. This current drives the membrane potential toward the K + equilibrium potential and thus affects input resistance. TREK1 displays low basal activity when expressed alone (1) but can be strongly stimulated by temperature (2), mechanical stretch (3), external alkalization (4), intracellular acidification (5), polyunsaturated fatty acids (PUFAs) (6), lysophospholipids (7), phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] (8, 9), and pharmacological agents such as volatile anesthetics (10) and riluzole (11). TREK1 is inhibited by neurotransmitters and hormones that activate Gq and Gs pathways (3,(12)(13)(14) and pharmacological agents such as the antidepressant drug fluoxetine (15). TREK1 gene inactivation produces mice with decreased sensitivity to volatile anesthetics, impaired PUFA-mediated neuroprotection (16), and altered perception of pain (17). These mice also display a depression-resistant phenotype (18). This phenotype is in agreement with the sensitivity of TREK1 to fluoxetine, a widely used antidepressant drug.In the last decade, much effort has been made to elucidate the gating mechanism of the TREK1 channel. The cytosolic carboxylterminal domain of TREK1 that follows its fourth transmembrane domain (post-M4) has been implicated in its function and regulation. A glutamate residue, E306, has been shown to be a key element for stimulation by intracellular acidification (19) and a cluster of basic residues in the same region has been shown to be involved in TREK1 regulation by phospholipid (8). However, the mechanism of regulation of the channel by Gq-coupled receptors (GqPCR) and by pharmacological agents such as fluoxetine remains unclear.I...

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

“…At the molecular level, an electrostatic interaction between phospholipids and the Ct sequence proximal to the fourth transmembrane domain of TREKs has been proposed to underlie the regulation of these proteins by PI(4,5)P 2 [5,31]. However, the strong inhibition of TREKs by anionic PI(4, 5)P 2 stands in apparent contradiction to the inhibitory effect of poly-L, which has the opposite charge.…”

“…In subsequent reviews, Chemin et al demonstrated that PI(4,5)P 2 inhibited TREK-1 in about half of i-o patches on COS-7 cells and hippocampal neurons [3,5]. Nevertheless, most relevant studies and reviews by other researchers regard PI(4, 5)P 2 as a stimulatory factor of TREKs [2,11,30,31,33].…”

Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels

“…For both TREK1 and TREK2, intracellular acidification increases the potassium current by protonating Glu residues on the post-M4 C-terminal domain (Ctd), which controls channel gating through interaction with the plasma membrane (6,26). Consistent with previous studies, acidifying the intracellular solution from internal pH (pH i ) 7.4 to pH i 5.5 increased TREK2 current (I TREK2 ) by 27 ± 5-fold, whereas I TREK1 showed only an 8 ± 3-fold increase (P < 0.01) (Fig.…”

“…They are not very active under basal conditions but can be dynamically stimulated by a wide range of stimuli, including mechanical stretch (1), heat (2,3), phospholipids (4)(5)(6), and polyunsaturated fatty acids (1,7). pH is an especially prominent regulator of these channels.…”

Diacylglycerol mediates regulation of TASK potassium channels by Gq-coupled receptors

“…Although its location close to the membrane's inner leaflet may suggest a role in DAG binding, the membraneproximal C terminus is implicated in gating of other K2P channels by a variety of stimuli. In TREK channels it mediates channel gating by phospholipids, membrane tension, intracellular pH and the antidepressant fluoxetine [45][46][47] . In TASK channels, the VLRFLT motif is also essential for channel activation by volatile anaesthetics 14 .…”