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

Abstract

Aims: The mode of action by which doxapram acts as a respiratory stimulant in humans is controversial. Studies in rodent models, have shown that doxapram is a more potent and selective inhibitor of TASK-1 and TASK-1/TASK-3 heterodimer channels, than TASK-3. Here we investigate the direct effect of doxapram and chirally separated, individual positive and negative enantiomers of the compound, on both human and mouse, homodimeric and heterodimeric variants of TASK-1 and TASK-3.

Methods: Whole-cell patch clamp electrophysiology on tsA201 cells was used to assess the potency of doxapram on cloned human or mouse TASK-1, TASK-3 and TASK-2 channels. Mutations of amino acids in the pore-lining region of TASK-3 channels were introduced using site-directed mutagenesis.

Results: Doxapram was an equipotent inhibitor of human TASK-1 and TASK-3 channels, compared with mouse channel variants, where it was more selective for TASK-1 and heterodimers of TASK-1 and TASK-3. The effect of doxapram could be attenuated by either the removal of the C-terminus of human TASK-3 channels or mutations of particular hydrophobic residues in the pore-lining region. These mutations, however, did not alter the effect of a known extracellular inhibitor of TASK-3, zinc. The positive enantiomer of doxapram, GAL-054, was a more potent antagonist of TASK channels, than doxapram, whereas the negative enantiomer, GAL-053, had little inhibitory effect.

Conclusion: These data show that in contrast to rodent channels, doxapram is a potent inhibitor of both TASK-1 and TASK-3 human channels, providing further understanding of the pharmacological profile of doxapram in humans and informing the development of new therapeutic agents.

Keywords: K2P channels; TASK-1 channels; TASK-3 channels; doxapram; enantiomers; heterodimers; respiratory stimulant.

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)

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

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)

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)

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

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 presence of 100 µM zinc (grey line)

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 compared with murTASK‐3 (data not shown). A concentration of 10 µM inhibited murTASK‐1 channels by 63% [95% CI = 61 to 65, n = 3] compared to an inhibition of 33% [95% CI = 19 to 46, n = 4] for murTASK‐3