Halogenated Ether, Alcohol, and Alkane Anesthetics Activate TASK-3 Tandem Pore Potassium Channels Likely through a Common Mechanism

Abstract

The TWIK-related acid-sensitive potassium channel 3 (TASK-3; KCNK9) tandem pore potassium channel function is activated by halogenated anesthetics through binding at a putative anesthetic-binding cavity. To understand the pharmacologic requirements for TASK-3 activation, we studied the concentration-response of TASK-3 to several anesthetics (isoflurane, desflurane, sevoflurane, halothane, -chloralose, 2,2,2-trichloroethanol [TCE], and chloral hydrate), to ethanol, and to a panel of halogenated methanes and alcohols. We used mutagenesis to probe the anesthetic-binding cavity as observed in a TASK-3 homology model. TASK-3 activation was quantified by Ussing chamber voltage clamp analysis. We mutagenized the residue Val-136, which lines the anesthetic-binding cavity, its flanking residues (132 to 140), and Leu-122, a pore-gating residue. The 2-halogenated ethanols activate wild-type TASK-3 with the following rank order efficacy (normalized current [95% confidence interval]): 2,2,2-tribromo-(267% [240-294])> 2,2,2-trichloro-(215% [196-234]) > chloral hydrate (165% [161-176]) > 2,2-dichloro- > 2-chloro ≈ 2,2,2-trifluoroethanol > ethanol. Similarly, carbon tetrabromide (296% [245-346]), carbon tetrachloride (180% [163-196]), and 1,1,1,3,3,3-hexafluoropropanol (200% [194-206]) activate TASK-3, whereas the larger carbon tetraiodide and -chloralose inhibit. Clinical agents activate TASK-3 with the following rank order efficacy: halothane (207% [202-212])> isoflurane (169% [161-176]) > sevoflurane (164% [150-177]) > desflurane (119% [109-129]). Mutations at and near residue-136 modify TCE activation of TASK-3, and interestingly M159W, V136E, and L122D were resistant to both isoflurane and TCE activation. TASK-3 function is activated by a multiple agents and requires a halogenated substituent between ∼30 and 232 cm/mol volume with potency increased by halogen polarizeability. Val-136 and adjacent residues may mediate anesthetic binding and stabilize an open state regulated by pore residue Leu-122. Isoflurane and TCE likely share commonalities in their mechanism of TASK-3 activation.

Effects of the ventilatory stimulant, doxapram on human TASK‐3 (KCNK9, K2P9.1) channels and TASK‐1 (KCNK3, K2P3.1) channels

“…Indeed, for known gain-of-function mutations on these channels and other related K2P channels, regulation by blockers or activators is modified when the link between the C-terminus and the selectivity filter is disrupted. 29 As well as for doxapram 22 the L239 residue has previously been identified in TASK-1 for its involvement in A1899 inhibition 20 and more recently in A293 inhibition. With the structure of the intracellular C-termini of K2P channels not determined by existing crystal structures, the involvement of this region remains difficult to evaluate.…”

“…[20][21][22]29 The key amino acids (AA) identified are Leucine (L) 122, Glycine (G) 236, L239 and Valine (V) 242, which are homologous in the human clones ( Figure 5A,B). [20][21][22]29 The key amino acids (AA) identified are Leucine (L) 122, Glycine (G) 236, L239 and Valine (V) 242, which are homologous in the human clones ( Figure 5A,B).…”

“…Evidence from molecular modelling, docking and aspartate scanning mutagenesis of rat TASK-1 and TASK-3, using inhibitory compounds such as A1899, PKTHPP and doxapram have suggested a common intracellular binding site, comprised of hydrophobic residues from the M2 and M4 transmembrane domains, within the intracellular pore region of these channels. [20][21][22]29 The key amino acids (AA) identified are Leucine (L) 122, Glycine (G) 236, L239 and Valine (V) 242, which are homologous in the human clones ( Figure 5A,B). In rat TASK-3 mutation of these specific AA to aspartate (D), considerably affected the efficacy of these compounds highlighting the importance of this region for the action of these particular compound types.…”

Molecular determinants of chemical modulation of two‐pore domain potassium channels

“…Noticeable, K2P crystal structures reported so far have divergent positions of the HRE‐like residues which are located lower on M4 helix (Figure b,c) making homology modeling tedious, but suggesting a putative divergence in the K2P family at this position (Bertaccini et al., ). More recently, it was revealed that another anesthetic, trichloroethanol (Figure ), activates TASK‐3 and may share a similar binding position in that particular interface (Luethy, Boghosian, Srikantha, & Cotten, ). Mutations of other putative pocket residues at positions 133, 136, and 140 reduce anesthetic activation, while at residue 139 enhances it, molecular modeling showing the residues are not in the close proximity of the HRE, being located upwards and defining a different binding pocket, placed directly behind the M2 helix.…”

“…Mutation L122D also impairs trichloroethanol activation of TASK‐3. As L122 lines the inner cavity and its corresponding one in TWIK‐1 (L146) is involved in hydrophobic gating (Aryal et al., ), TASK‐activating anesthetics binding at the M2/M3/M4 interface could allosterically transduce their effects to the pore to deactivate a hydrophobic gating effect (Luethy et al., ). TASK channels are also blocked by the anesthetic bupivacaine (Figure ), in a distinct binding site, but the molecular features of such blockade, reported very recently (Rinné et al., ), have important implications and will be presented later in the manuscript.…”