Journal: bioRxiv
Article Title: CryoEM Structure of the human THIK-1 K2P K + Channel Reveals a Lower ‘Y-gate’ Regulated by Lipids and Anaesthetics
doi: 10.1101/2024.06.26.600475
Figure Lengend Snippet: a, Representative recording at −80 mV from an inside-out patch containing WT THIK-1 channels with symmetrical K + concentrations (120 mM [K + ] ex. /120 mM [K + ] int. ) at pH 7.4. Channel currents were inhibited dose-dependently with increasing concentrations of halothane applied to the intracellular side of the membrane. Note, halothane effects can be washed and recovered and the currents inhibited with TPenA. b, Analysis of halothane inhibition for THIK-1 WT from recordings as in panel a in the absence (gray) and presence (orange) of 0.5 mM TPenA which produces ∼ 80 % block of initial currents. c, Analysis of halothane inhibition from recordings as in panel a for WT THIK-1 and indicated mutant channels. d, THIK-1 with docked halothane in the vestibule. Residues in close proximity are highlighted as sticks. For clarity, residues 121-138 in M2’ as well as PH2’ are not shown. e, Comparison of the structures of halothane, isoflurane and sevoflurane. f, Analysis of isoflurane inhibition of WT THIK-1 and channel mutants (i.e., Y273A and T237A). g, Summary of volatile anaesthetic inhibition with either 3.0 mM halothane, 4.9 mM isoflurane and 0.24 mM sevoflurane for WT THIK-1 and mutant channels as indicated. h, Representative recording under conditions as in panel a showing dose-dependent halothane inhibition for THIK-1 channels activated with 5.0 µM oleoyl-CoA. i , Analysis of halothane inhibition in the absence and presence of 5.0 µM oleoyl-CoA from recordings as in panels a and h. j, Fold activation of WT THIK-1 with 5.0 µM oleoyl-CoA in the absence and presence of 15.2 mM halothane. Values are given as mean ± s.e.m with number (n) of individual recordings indicated above the bars.
Article Snippet: Tetrapentylammonium (TPenA), linoleic acid, L-α-PI(4,5)P 2 (brain PI(4,5)P 2 , PIP 2 ) (Sigma-Aldrich/Merck, Germany) and oleoyl-CoA (LC-CoA 18:1) (Avanti Polar Lipids, USA) were prepared as stocks (1 - 100 mM) in DMSO, stored at −80 °C and diluted to the final concentration in the intracellular recording solution.Halothane 99 % (Sigma-Aldrich/Merck, Germany), isoflurane (Baxter, Germany) and sevoflurane 100 % (AbbVie, Germany), respectively were added to the intracellular recording solution, shaken for 3 min. and after clear phase separation (∼ 5 min.) the intracellular solution was used for experiments within 15 min.
Techniques: Membrane, Inhibition, Blocking Assay, Mutagenesis, Comparison, Activation Assay
![a, The Y-gate viewed from the side (M2’ hidden for clarity), and from the bottom, showing residue I139 in the same horizontal level as Y273 and also part of the constriction formed by the Y-gate. b, Relative whole-cell current amplitudes of WT THIK-1 and channels with mutations in the Y-gate. All currents are normalised to WT. c, Cell-attached recordings of 1s duration at ± 200mV containing single WT, I139S and Y273S THIK-1 channels, as indicated. The closed (c) and open channel (o) levels are indicated. d, Comparison of single-channel open probability ( P o ) and single channel current amplitude ( i ) of WT, I139S and Y273S THIK-1 channels in cell-attached patches at −200mV. Numbers above the bars denote the number of experiments. e, Representative recording at −80 mV from an inside-out patch containing WT THIK-1 channels with symmetrical K + concentrations (120 mM [K + ]) at pH 7.4. Channel currents were activated with 5 µM oleoyl-CoA) applied to the intracellular side of the membrane and then inhibited dose-dependently with <t>TPenA</t> as indicated. Inlay shows equal inhibition with TPenA at the unstimulated basal state of the channels. f,g, Analysis of the apparent affinity for TPenA in the indicated states from recordings in panel e showing increased TPenA sensitivity after lipid activation. h, Analysis of TPenA kinetics for block and release of WT THIK-1 in unstimulated and lipid-activated states. i - j, Analysis of apparent TPenA affinity ( i ) and kinetics ( j ) for WT and indicated THIK-1 mutants from recordings as in the inlay in panel e. All values are given as mean ± s.e.m with number (n) of individual recordings indicated above the bars.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_75/10__1101_slash_2024__06__26__600475/10__1101_slash_2024__06__26__600475___F2.large.jpg)
![a, Representative recording at −80 mV from an inside-out patch containing WT THIK-1 channels with symmetrical K + concentrations (120 mM [K + ] ex. /120 mM [K + ] int. ) at pH 7.4. Channel currents were inhibited dose-dependently with increasing concentrations of halothane applied to the intracellular side of the membrane. Note, halothane effects can be washed and recovered and the currents inhibited with TPenA. b, Analysis of halothane inhibition for THIK-1 WT from recordings as in panel a in the absence (gray) and presence (orange) of 0.5 mM TPenA which produces ∼ 80 % block of initial currents. c, Analysis of halothane inhibition from recordings as in panel a for WT THIK-1 and indicated mutant channels. d, THIK-1 with docked halothane in the vestibule. Residues in close proximity are highlighted as sticks. For clarity, residues 121-138 in M2’ as well as PH2’ are not shown. e, Comparison of the structures of halothane, isoflurane and sevoflurane. f, Analysis of isoflurane inhibition of WT THIK-1 and channel mutants (i.e., Y273A and T237A). g, Summary of volatile anaesthetic inhibition with either 3.0 mM halothane, 4.9 mM isoflurane and 0.24 mM sevoflurane for WT THIK-1 and mutant channels as indicated. h, Representative recording under conditions as in panel a showing dose-dependent halothane inhibition for THIK-1 channels activated with 5.0 µM oleoyl-CoA. i , Analysis of halothane inhibition in the absence and presence of 5.0 µM oleoyl-CoA from recordings as in panels a and h. j, Fold activation of WT THIK-1 with 5.0 µM oleoyl-CoA in the absence and presence of 15.2 mM halothane. Values are given as mean ± s.e.m with number (n) of individual recordings indicated above the bars.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_75/10__1101_slash_2024__06__26__600475/10__1101_slash_2024__06__26__600475___F3.large.jpg)