trimethylammonium ethyl methanethiosulfonate bromide mts et  (Toronto Research Chemicals)

Journal: bioRxiv

Article Title: Ion occupancy of the selectivity filter controls opening of a cytoplasmic gate in the K2P channel TALK-2

doi: 10.1101/2023.11.22.568211

Figure Lengend Snippet: a Representative current trace measured under voltage-clamp conditions at +40 mV from inside-out patches of Xenopus laevis oocytes expressing WT TALK-2 channels symmetrical K + (120 mM [K + ] ex. /120 mM [K + ] int. ) at pH 7.4. Channel currents were activated with the indicated compounds (1 mM 2-APB and 5 µM oleoyl-CoA) applied to the intracellular side of the membrane. Inlays showing current-voltage responses of 2-APB (blue) and oleoyl-CoA (green) activation compared to the basal state (black) by the indicated voltage protocol. b Pore homology model of TALK-2 based on the crystal structure of TASK-1 (PDB ID: 6RV3, chains A, B) with the SF highlighted in red, K + ions in black, and cysteine residues (L145C and Q266C) for MTS-ET modification in yellow. c Pore cavity zoom-in. Cut outs display the localization of L145C in the inner cavity and Q266C at the intracellular end of the pore. d Representative measurements of TALK-2 L145C channels showing state-dependent MTS-ET modification with no effect under unstimulated (basal) conditions or inhibition upon application of 1.0 mM MTS-ET in pre-activated states with 1.0 mM 2-APB (blue) or 5.0 µM oleoyl-CoA (green) with the indicated time constants (τ), respectively. e Same measurements as in (d) with TALK-2 Q266C channels showing state-independent modification with activation upon application of 1.0 mM MTS-ET. f Current responses recorded with the indicated voltage protocol in symmetrical K + showing activation of WT TALK-2 with increasing 2-APB concentrations. The dotted line shows the increase and saturation of current amplitudes with 2-APB at + 40 mV. g 2-APB dose-response curves analyzed from measurements as in (f) for WT TALK-2 (blue), TALK-2 L145C (black) and WT TREK-1 (gray) channels. h Correlation between the fold change in current amplitudes of TALK-2 L145C channels at +40 mV (black squares) and the rate of MTS-ET modification (1/τ) at +40 mV (orange squares) with different 2-APB concentrations. Values are given as mean ± s.e.m with number (n) of experiments indicated in the figure and supplementary tables 1 and 2.

Article Snippet: Tetra-pentyl-ammonium chloride (TPenA), 2-aminoethoxydiphenyl borate (2-APB) (Sigma-Aldrich/Merck, Germany), BL-1249, A1899 (Tocris Bioscience, Germany), AVE0118, S9947 (Axon Medchem, 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. (2-(Trimethylammonium)ethyl) MethaneThioSulfonate Chloride (MTS-ET) (Toronto Research Chemicals, USA) was directly dissolved to the desired concentration of 1.0 mM in the intracellular recording solution prior to each experiment.

Techniques: Expressing, Membrane, Activation Assay, Modification, Inhibition

Journal: bioRxiv

Article Title: Ion occupancy of the selectivity filter controls opening of a cytoplasmic gate in the K2P channel TALK-2

doi: 10.1101/2023.11.22.568211

Figure Lengend Snippet: a Representative measurement of WT TALK-2 channels from an inside-out patch in symmetrical K + at +40 mV with increasing 2-APB concentrations (c 1 - c 5 ) applied from the intracellular side at indicated time points (blue arrows). At steady-state current levels with 2-APB intracellular K + was exchanged by Rb + showing an enhanced activatory Rb + ion effect on the SF in 2-APB pre-activated channels. b Same recording as in (a) for WT TREK-1 K 2P channels showing the stepwise loss of Rb + activation in the presence of increasing 2-APB concentrations. c Correlation of Rb + -induced currents from measurements as in (a,b) in the presence of indicated 2-APB concentrations for either WT TALK-2 (black) or WT TREK-1 (gray) channels. d Gating scheme highlighting the effect of 2-APB and Rb + on the lower and selectivity filter gate in TALK-2 channels. Values are given as mean ± s.e.m with number (n) of experiments indicated in the figure.

Article Snippet: Tetra-pentyl-ammonium chloride (TPenA), 2-aminoethoxydiphenyl borate (2-APB) (Sigma-Aldrich/Merck, Germany), BL-1249, A1899 (Tocris Bioscience, Germany), AVE0118, S9947 (Axon Medchem, 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. (2-(Trimethylammonium)ethyl) MethaneThioSulfonate Chloride (MTS-ET) (Toronto Research Chemicals, USA) was directly dissolved to the desired concentration of 1.0 mM in the intracellular recording solution prior to each experiment.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Ion occupancy of the selectivity filter controls opening of a cytoplasmic gate in the K2P channel TALK-2

doi: 10.1101/2023.11.22.568211

Figure Lengend Snippet: a TALK-2 current responses to voltage families as indicated in symmetrical K + (120 mM [K + ] ex. /120 mM [K + ] int. ) at pH 7.4 on both sides (black traces) and at extracellular pH 9.5 (brown traces). b Cartoon illustrating a simple TALK-2 channel gating model and pore accessibility to MTS-ET by alterations of the pH e that directly affects the SF. c Representative modification and subsequent irreversible inhibition with 1.0 mM MTS-ET of TALK-2 L145C channels pre-activated by extracellular alkalinization (pH e 9.5). d TALK-2 channel currents with intracellular Rb + (120 mM [K + ] ex. /120 mM [Rb + ] int. ) at pH 7.4 for different potentials as indicated showing a maximum P O reached for potentials positive to ∼+135 mV (V max ), as further depolarizations do not increase the tail current amplitudes. e Voltage activation (conductance-voltage (G-V) curves) with V 1/2 values of 72 ± 2 mV and 66 ± 3 mV of WT TALK-2 and L145C mutant channels, respectively. f Cartoon of a simple gating model with Rb + as an amplifier for voltage activation targeting the SF and subsequently the lower gate in TALK-2 channels. g , h Representative measurements at +40 mV of WT (g) and L145C mutant TALK-2 channels (h) showing a non-modifiable state or almost complete modification/inhibition with 1.0 mM MTS-ET within 60 s in intracellular Rb + , respectively. i Correlation between the fold change of tail current amplitudes (black squares) of TALK-2 L145C channels and the incidental rate of MTS-ET modification (1/τ) (orange squares) with intracellular Rb + at different potentials as indicated. Values are given as mean ± s.e.m with the number (n) of experiments indicated in the figure and supplementary table 1 - 3.

Article Snippet: Tetra-pentyl-ammonium chloride (TPenA), 2-aminoethoxydiphenyl borate (2-APB) (Sigma-Aldrich/Merck, Germany), BL-1249, A1899 (Tocris Bioscience, Germany), AVE0118, S9947 (Axon Medchem, 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. (2-(Trimethylammonium)ethyl) MethaneThioSulfonate Chloride (MTS-ET) (Toronto Research Chemicals, USA) was directly dissolved to the desired concentration of 1.0 mM in the intracellular recording solution prior to each experiment.

Techniques: Modification, Inhibition, Activation Assay, Mutagenesis

Journal: bioRxiv

Article Title: Ion occupancy of the selectivity filter controls opening of a cytoplasmic gate in the K2P channel TALK-2

doi: 10.1101/2023.11.22.568211

Figure Lengend Snippet: a Current responses measured in inside-out patches under voltage-clamp conditions from Xenopus laevis oocytes expressing WT TALK-2 channels activated with indicated voltage steps under symmetrical ion conditions (120 mM [K + ] ex. /120 mM [X + ] int. ) at pH 7.4 with either intracellular K + (black trace, basal state) or Rb + (red trace, activated state) or with 1.0 mM TPenA in Rb + (orange trace). Note, the presence of TPenA shows slowing of deactivation resulting in a tail current cross-over (zoom-in). Cartoon depicting a simple model for TALK-2 channel gating, whereby Rb + activation of the SF enables blocker (e.g., TPenA) binding in the pore cavity and unbinding facilitates lower and SF gate closure at −80 mV. b Same recording as in (a) with TREK-2 K 2P channels showing inhibition with 100 µM TPenA without tail current cross-over. c Representative current responses of WT TALK-2 channels to a 300 ms voltage step as indicated in the absence (black traces) and presence of 1.0 mM TPenA (orange traces) applied to the intracellular side of the membrane. d Representative measurement of TALK-2 channel currents at +40 mV showing dose-dependent TPenA inhibition in the pre-activated state with 1.0 mM 2-APB. e Dose-response curves of TPenA inhibition from measurements as in (d) for TALK-2 in unstimulated (basal) conditions (black) and pre-activated states with 2-APB (blue) with altering apparent affinities for TPenA (IC 50 (0.2 mM 2-APB) = 778 ± 116, IC 50 (0.5 mM 2-APB) = 215 ± 28, IC 50 (1.0 mM 2-APB) = 54 ± 10). f Residual currents of WT and L264A mutant TALK-2 channels at +40 mV upon block of 1.0 mM TPenA at the indicated conditions. g Residual currents of unstimulated (black), 2-APB pre-activated WT (blue) and L264A mutant (gray) TALK-2 channels after inhibition with the indicated open channel blocker. h Simplified gating scheme indicating that blocker interact with the open state of TALK-2 to produce inhibition. Values are given as mean ± s.e.m with the number (n) of experiments indicated in the figure.

Article Snippet: Tetra-pentyl-ammonium chloride (TPenA), 2-aminoethoxydiphenyl borate (2-APB) (Sigma-Aldrich/Merck, Germany), BL-1249, A1899 (Tocris Bioscience, Germany), AVE0118, S9947 (Axon Medchem, 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. (2-(Trimethylammonium)ethyl) MethaneThioSulfonate Chloride (MTS-ET) (Toronto Research Chemicals, USA) was directly dissolved to the desired concentration of 1.0 mM in the intracellular recording solution prior to each experiment.

Techniques: Expressing, Activation Assay, Binding Assay, Inhibition, Membrane, Mutagenesis, Blocking Assay

Journal: bioRxiv

Article Title: Ion occupancy of the selectivity filter controls opening of a cytoplasmic gate in the K2P channel TALK-2

doi: 10.1101/2023.11.22.568211

Figure Lengend Snippet: a, b G-V curves analyzed from tail currents at −80 mV after 300 ms pre-pulse steps (−120 mV to +160 mV with 5 mV increments) under symmetrical ion conditions with intracellular Rb + of WT and mutant TALK-2 channels as indicated (b) and the summary of V 1/2 values from Boltzmann fits to the corresponding G-V curves (a). c Correlation of the V 1/2 shifts of mutant TALK-2 channels at basal and WT TALK-2 channels at indicated conditions with the time constants of modification of TALK-2 L145C channels under the corresponding activatory conditions or in combination with the respective g-o-f mutation. d Simplified energetic scheme depicting the electrical work (ΔG = zFΔV 1/2 ) required to open both gates (ΔG total ) with the individual contribution of the SF gate (ΔG SF ) and lower gate (ΔG LG ). Mutations (as indicated in the inlay) that open the lower gate reduced this electrical work as seen in the positive V 1/2 shifts of the G-V curve. Note, our results actually show that both gates are strongly positively coupled and, thus, the pre-open state (just the lower gate open) is just a conceptual state to illustrate the energetic contribution of the lower gate. Values are given as mean ± s.e.m with the number (n) of experiments indicated in the figure and supplementary table 3 and 4 (n.d. = not determinable, n.-e. = non-expressing).

Article Snippet: Tetra-pentyl-ammonium chloride (TPenA), 2-aminoethoxydiphenyl borate (2-APB) (Sigma-Aldrich/Merck, Germany), BL-1249, A1899 (Tocris Bioscience, Germany), AVE0118, S9947 (Axon Medchem, 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. (2-(Trimethylammonium)ethyl) MethaneThioSulfonate Chloride (MTS-ET) (Toronto Research Chemicals, USA) was directly dissolved to the desired concentration of 1.0 mM in the intracellular recording solution prior to each experiment.

Techniques: Mutagenesis, Modification, Expressing

Journal: bioRxiv

Article Title: Ion occupancy of the selectivity filter controls opening of a cytoplasmic gate in the K2P channel TALK-2

doi: 10.1101/2023.11.22.568211

Figure Lengend Snippet: a-c G-V curves analyzed from current-voltage families (−120 mV to +160 mV with 5 mV increments) measured under symmetrical ion conditions with intracellular Rb + of WT TALK-2 (a), WT TREK-2 (b) and L264A mutant TALK-2 channels (c) in the absence (black traces) and presence of 0.1 mM (brown trace) or 1.0 mM TPenA (orange traces), respectively. d G-V curves analyzed from WT TALK-2 tail currents in the presence of pH e 7.4 (black trace, 9.0 (orange trace) and 10.5 (brown trace). e V 1/2 values from G-V curves analyzed as in (d) with varying pH e as indicated. f V 1/2 values from G-V curves of WT TALK-2 channels activated with 1.0 mM 2-APB or 5.0 µM oleoyl-CoA. g Normalized currents from TEVC measurements of Xenopus oocytes expressing WT and mutant (L262A or L264A respectively) TALK-2 channels activated by increasing pH e from 5.5 to 10.5 with 0.5 pH increments. Currents were elucidated with a voltage protocol ramped from −120 mV to +45 mV within 3.5 s, analyzed at +40 mV and normalized to pH 10.5. Values are given as mean ± s.e.m with the number (n) of experiments indicated in the figure and supplementary table 3 and 4 (n.d. = not determinable, n.-e. = non-expressing).

Article Snippet: Tetra-pentyl-ammonium chloride (TPenA), 2-aminoethoxydiphenyl borate (2-APB) (Sigma-Aldrich/Merck, Germany), BL-1249, A1899 (Tocris Bioscience, Germany), AVE0118, S9947 (Axon Medchem, 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. (2-(Trimethylammonium)ethyl) MethaneThioSulfonate Chloride (MTS-ET) (Toronto Research Chemicals, USA) was directly dissolved to the desired concentration of 1.0 mM in the intracellular recording solution prior to each experiment.

Techniques: Mutagenesis, Expressing