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

Figures

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  Figure 1. Effect of doxapram on human cloned TASK‐1, TASK‐3 and TASK‐2 channels. (A) Concentration‐response curve for doxapram inhibition of human (h) TASK‐1 current. (B) hTASK‐1 currents evoked by ramp changes in voltage in control conditions and in the presence of doxapram over a range of concentrations (0.3‐100 µM). (C) Concentration‐response curve for doxapram inhibition of hTASK‐3 current. (D) hTASK‐3 currents evoked by ramp changes in voltage in control conditions and in the presence of doxapram over a range of concentrations (0.3‐100 µM). (E) A plot of % inhibition by 10 µM doxapram from individual cells expressing either hTASK‐1, hTASK‐3 or hTASK‐2 human cDNA. Error bars represent the 95% CI. *P < .05, ****P < .0001; One‐way ANOVA, followed by a Dunnett's multiple comparisons test. (F) Time course plot showing the acute application of 10 µM Doxapram (blue line) on hTASK‐2 current. Each point is a 5 millisecond (ms) average of the difference current between that at −40 mV and that at −80 mV (see methods for detailed description of voltage‐ramp protocol)

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  Figure 2. Effect of doxapram on mouse cloned TASK‐1, TASK‐3 homodimeric and forced heterodimeric channels. (A) Concentration‐response curve for doxapram inhibition of murTASK‐1 current over a range of concentrations (0.3‐100 µM). (B) Concentration‐response curve for doxapram inhibition of murTASK‐3 current over a range of concentrations (10‐300 µM). (C) A plot of % inhibition by 10 µM doxapram from individual cells expressing either homodimeric murTASK‐3, homodimeric murT1, heterodimeric murTASK‐3_murTASK‐1 and heterodimeric murTASK‐1_murTASK‐3 cDNA. Error bars represent the 95% CI. (D) Raw data trace from exemplar mouse heterodimeric murTASK‐1_murTASK‐3 in control (black line) and 10 µM doxapram (blue line) using a step‐ramp voltage protocol as detailed in the Methods

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  Figure 3. Removal of the carboxy terminal of murTASK‐3 (murT3) attenuates doxapram effect further. (A) Cartoon to depict the amino acid structure of the carboxy terminal of murT3, the location of putative phosphorylation sites and the introduction of the stop codon. (B) Box and Whiskers plot of doxapram (10 µM) inhibition of murT3_Δ250 and murT3 wild type. Bars represent the min and max inhibition for each channel type. (C) murT3_ Δ250 currents evoked by ramp changes in voltage from −120 to + 20 mV in control conditions (black line) and in the presence of 10 µM doxapram (blue line)

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  Figure 4. Removal of the carboxy terminal of human TASK‐3 attenuates doxapram effect. (A) Cartoon to compare the identity of the amino acid structure of the carboxy terminal of human TASK‐3 and human TASK‐1. (B) Box and Whiskers plot of doxapram (10 µM) inhibition of hTASK‐3_Δ250 and WT hTASK‐3. Bars represent the min and max inhibition for each channel type. (C) hTASK‐3_ Δ250 currents evoked by ramp changes in voltage from −120 to + 20 mV in control conditions (black line) and in the presence of 10 µM doxapram (blue line)

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  Figure 5. Computer homology model of human TASK‐3 channel with indicated putative binding site. (A) Homology model of human TASK‐3 channel based upon TRAAK crystal structure (PDB ID 3UM7)54 depicting location of the four amino acids (AA) that form the putative site, L122, G236, L239 and V242 (shown with arrows) as viewed from beneath the channel. (B) AA sequence alignment of human TASK‐1 and TASK‐3. Dashes represent gaps in the sequence and numbers represent the position of the starting AA. The black box highlights the AA’s that form the putative site

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  Figure 6. Pore‐lining residues of the M2 and M4 domains are influenential for doxapram inhibition of the human TASK‐3 channel. (A) graph of current density (pA pF‐1) measured from individual cells transiently expressing WT TASK‐3, TASK‐3_L122D, TASK‐3_G236D, TASK‐3_L239D, TASK‐3_V242D. Error bars represent the 95% CI and * statistical significance (*P < .05; ****P < .0001). (B) A plot of zero current level (mV) measured from individual cells transiently expressing WT TASK‐3, TASK‐3_L122D, TASK‐3_G236D, TASK‐3_L239D, TASK‐3_V242D. Error bars represent the 95% CI. (C) Box and Whiskers plot of doxapram (10 µM) inhibition of WT TASK‐3, TASK‐3_L122D, TASK‐3_G236D, TASK‐3_L239D and TASK‐3_V242D. Bars represent the min and max inhibition for each channel type. (D) hT3_ L122D currents evoked by ramp changes in voltage from −120 to + 20 mV in control conditions (black line) and in the presence of 10 µM doxapram (blue line). (E) Box and Whiskers plot of zinc (100 µM) inhibition of WT TASK‐3 and TASK‐3_L122D. Bars represent the min and max inhibition for each channel type. (F) hT3_ L122D currents evoked by ramp changes in voltage from −120 to + 20 mV in control conditions (black line) and in the…

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  Figure 7. The (+)—enantiomer (GAL‐054) is responsible for the inhibitory effects observed with doxapram on human TASK channels. (A) A plot of % inhibition of 1, 3 and 10 µM GAL‐054 on human TASK‐1 channels. Each point represents an individual cell and the error bars represent the 95% CI. (B) human TASK‐1 currents evoked by ramp changes in voltage from −120 to + 20 mV in control conditions (black line) and in the presence of 10 µM GAL‐054 (red line). (C) A plot of % inhibition of 1, 3 and 10 µM GAL‐054 on human TASK‐3 channels. (D) human TASK‐3 currents in control conditions (black line) and in the presence of 10 µM GAL‐054 (red line). (E) A plot of % inhibition of 10, 100 and 300 µM GAL‐053 on human TASK‐1 channels. (F) human TASK‐1 currents in control conditions (black line) and in the presence of 10 µM GAL‐053 (green line). (G) A plot of % inhibition of 10, 100 and 300 µM GAL‐053 on human TASK‐3 channels. (H) human TASK‐3 currents in control conditions (black line) and in the presence of 10 µM GAL‐053 (green line). The specificity observed with mouse TASK channels remained unchanged with for the (+)—enantiomer GAL‐054, with the enantiomer being twice as potent on murTASK‐1 channels…