Convergence of Multiple Stimuli to a Single Gate in TREK1 and TRAAK Potassium Channels.
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ABSTRACT:
SUBMITTER: Choveau FS
PROVIDER: S-EPMC8514629 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mechanosensitive ion channels underlie neuronal responses to physical forces in the sensation of touch, hearing, and other mechanical stimuli. The fundamental basis of force transduction in eukaryotic mechanosensitive ion channels is unknown. Are mechanical forces transmitted directly from membrane to channel as in prokaryotic mechanosensors or are they mediated through macromolecular tethers attached to the channel? Here we show in cells that the K(+) channel TRAAK (K2P4.1) is responsive to mechanical forces similar to the ion channel Piezo1 and that mechanical activation of TRAAK can electrically counter Piezo1 activation. We then show that the biophysical origins of force transduction in TRAAK and TREK1 (K2P2.1) two-pore domain K(+) (K2P) channels come from the lipid membrane, not from attached tethers. These findings extend the "force-from-lipid" principle established for prokaryotic mechanosensitive channels MscL and MscS to these eukaryotic mechanosensitive K(+) channels.
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Project description:Mechanosensitive K(+) channels TREK1 and TREK2 form a subclass of two P-domain K(+) channels. They are potently activated by polyunsaturated fatty acids and are involved in neuroprotection, anesthesia, and pain perception. Here, we show that acidification of the extracellular medium strongly inhibits TREK1 with an apparent pK near to 7.4 corresponding to the physiological pH. The all-or-none effect of pH variation is steep and is observed within one pH unit. TREK2 is not inhibited but activated by acidification within the same range of pH, despite its close homology with TREK1. A single conserved residue, H126 in TREK1 and H151 in TREK2, is involved in proton sensing. This histidine is located in the M1P1 extracellular loop preceding the first P domain. The differential effect of acidification, that is, activation for TREK2 and inhibition for TREK1, involves other residues located in the P2M4 loop, linking the second P domain and the fourth membrane-spanning segment. Structural modeling of TREK1 and TREK2 and site-directed mutagenesis strongly suggest that attraction or repulsion between the protonated side chain of histidine and closely located negatively or positively charged residues in P2M4 control outer gating of these channels. The differential sensitivity of TREK1 and TREK2 to external pH variations discriminates between these two K(+) channels that otherwise share the same regulations by physical and chemical stimuli, and by hormones and neurotransmitters.
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Project description:Ion transport across cell membranes is essential to cell communication and signaling. Passive ion transport is mediated by ion channels, membrane proteins that create ion conducting pores across cell membrane to allow ion flux down electrochemical gradient. Under physiological conditions, majority of ion channel pores are not constitutively open. Instead, structural region(s) within these pores breaks the continuity of the aqueous ion pathway, thereby serves as activation gate(s) to control ions flow in and out. To achieve spatially and temporally regulated ion flux in cells, many ion channels have evolved sensors to detect various environmental stimuli or the metabolic states of the cell and trigger global conformational changes, thereby dynamically operate the opening and closing of their activation gate. The sensors of ion channels can be broadly categorized as chemical sensors and physical sensors to respond to chemical (such as neural transmitters, nucleotides and ions) and physical (such as voltage, mechanical force and temperature) signals, respectively. With the rapidly growing structural and functional information of different types of ion channels, it is now critical to understand how ion channel sensors dynamically control their gates at molecular and atomic level. The voltage and Ca(2+) activated BK channels, a K(+) channel with an electrical sensor and multiple chemical sensors, provide a unique model system for us to understand how physical and chemical energy synergistically operate its activation gate.
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Project description:Inactivation is an inherent property of most voltage-gated K(+) channels. While fast N-type inactivation has been analyzed in biophysical and structural details, the mechanisms underlying slow inactivation are yet poorly understood. Here, we characterized a slow inactivation mechanism in various KCNQ1 pore mutants, including L273F, which hinders entry of external Ba(2+) to its deep site in the pore and traps it by slowing its egress. Kinetic studies, molecular modeling, and dynamics simulations suggest that this slow inactivation involves conformational changes that converge to the outer carbonyl ring of the selectivity filter, where the backbone becomes less flexible. This mechanism involves acceleration of inactivation kinetics and enhancement of Ba(2+) trapping at elevated external K(+) concentrations. Hence, KCNQ1 slow inactivation considerably differs from C-type inactivation where vacation of K(+) from the filter was invoked. We suggest that trapping of K(+) at s(1) due to filter rigidity and hindrance of the dehydration-resolvation transition underlie the slow inactivation of KCNQ1 pore mutants.
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Project description:The selectivity filter of K(+) channels is conserved throughout all kingdoms of life. Carbonyl groups of highly conserved amino acids point toward the lumen to act as surrogates for the water molecules of K(+) hydration. Ion conductivity is abrogated if some of these carbonyl groups flip out of the lumen, which happens (i) in the process of C-type inactivation or (ii) during filter collapse in the absence of K(+). Here, we show that K(+) channels remain permeable to water, even after entering such an electrically silent conformation. We reconstituted fluorescently labeled and constitutively open mutants of the bacterial K(+) channel KcsA into lipid vesicles that were either C-type inactivating or noninactivating. Fluorescence correlation spectroscopy allowed us to count both the number of proteoliposomes and the number of protein-containing micelles after solubilization, providing the number of reconstituted channels per proteoliposome. Quantification of the per-channel increment in proteoliposome water permeability with the aid of stopped-flow experiments yielded a unitary water permeability pf of (6.9 ± 0.6) × 10(-13) cm(3)⋅s(-1) for both mutants. "Collapse" of the selectivity filter upon K(+) removal did not alter pf and was fully reversible, as demonstrated by current measurements through planar bilayers in a K(+)-containing medium to which K(+)-free proteoliposomes were fused. Water flow through KcsA is halved by 200 mM K(+) in the aqueous solution, which indicates an effective K(+) dissociation constant in that range for a singly occupied channel. This questions the widely accepted hypothesis that multiple K(+) ions in the selectivity filter act to mutually destabilize binding.
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Project description:Background and purposePharmacological separation of the background potassium currents of closely related K2P channels is a challenging problem. We previously demonstrated that ruthenium red (RR) inhibits TASK-3 (K2 P 9.1), but not TASK-1 (K2 P 3.1) channels. RR has been extensively used to distinguish between TASK currents in native cells. In the present study, we systematically investigate the RR sensitivity of a more comprehensive set of K2 P channels.Experimental approachK(+) currents were measured by two-electrode voltage clamp in Xenopus oocytes and by whole-cell patch clamp in mouse dorsal root ganglion (DRG) neurons.Key resultsRR differentiates between two closely related members of the TREK subfamily. TREK-2 (K2 P 10.1) proved to be highly sensitive to RR (IC50 = 0.2 μM), whereas TREK-1 (K2 P 2.1) was not affected by the compound. We identified aspartate 135 (D135) as the target of the inhibitor in mouse TREK-2c. D135 lines the wall of the extracellular ion pathway (EIP), a tunnel structure through the extracellular cap characteristic for K2 P channels. TREK-1 contains isoleucine in the corresponding position. The mutation of this isoleucine (I110D) rendered TREK-1 sensitive to RR. The third member of the TREK subfamily, TRAAK (K2 P 4.1) was more potently inhibited by ruthenium violet, a contaminant in some RR preparations, than by RR. DRG neurons predominantly express TREK-2 and RR-resistant TREK-1 and TRESK (K2 P 18.1) background K(+) channels. We detected the RR-sensitive leak K(+) current component in DRG neurons.Conclusions and implicationsWe propose that RR may be useful for distinguishing TREK-2 (K2P 10.1) from TREK-1 (K2P 2.1) and other RR-resistant K2 P channels in native cells.
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Project description:TRAAK is an ion channel from the two-pore domain potassium (K2P) channel family with roles in maintaining the resting membrane potential and fast action potential conduction. Regulated by a wide range of physical and chemical stimuli, the affinity and selectivity of K2P4.1 toward lipids remains poorly understood. Here we show the two isoforms of K2P4.1 have distinct binding preferences for lipids dependent on acyl chain length and position on the glycerol backbone. The channel can also discriminate the fatty acid linkage at the SN1 position. Of the 33 lipids interrogated using native mass spectrometry, phosphatidic acid had the lowest equilibrium dissociation constants for both isoforms of K2P4.1. Liposome potassium flux assays with K2P4.1 reconstituted in defined lipid environments show that those containing phosphatidic acid activate the channel in a dose-dependent fashion. Our results begin to define the molecular requirements for the specific binding of lipids to K2P4.1.
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