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| 083P London, UK Pharmacology 2016 |
The ventilatory stimulant, doxapram, is an equally potent inhibitor of the human two-pore domain potassium (K2P) channels, TASK-3 (KCNK9) and TASK-1 (KCNK3)
Introduction: Doxapram is used clinically in drug-induced ventilatory depression, chronic obstructive pulmonary disorder and apnoea (1). Its mechanism of action is controversial as animal and human studies are conflicting. It is proposed that doxapram stimulates peripheral chemoreceptors in the carotid bodies by inhibiting potassium channels, in particular TASK-1 and TASK-3 K2P channels (2). The aim of this study is to clarify the mechanism of action of doxapram on TASK channels.
Method: Currents through wildtype (WT) and mutated human and murine K2P channels transiently expressed in tsA201 cells were measured using whole-cell patch-clamp electrophysiology. Data are given as mean±SEM (n=cells) and statistical analysis used one-way ANOVA with post-hoc Dunnett’s multiple comparison test.
Results: Human TASK-3, TASK-1 and heterodimer channels were equally sensitive to block by doxapram (10µM throughout, 57±2%, n=8; 55±4%, n=8; 49±4%, n=6), respectively, p>0.05), despite this compound being suggested as a relatively selective rat TASK-1 inhibitor (3). In contrast, for mouse homologues, doxapram was a more potent inhibitor of TASK-1 than TASK-3 (63±5%, n=4 versus 22±5%, n=4). Several, identified TASK-3 residues have been suggested to contribute to an intracellular binding site for doxapram (4). Inhibition of human TASK-3 by doxapram was significantly attenuated by mutation of these residues, with least inhibition observed for L122D (8±2%, n=5) followed by L239D<G236D≤V242D (13±3%, n=8; 26±5%, n=8; 27±5%, n=5; p<0.05). In contrast, zinc (100µM) inhibition of TASK-3 (5) was only significantly affected by L122D (55±5%, n=7 versus 71±6%, n=6 for WT p<0.05). Substitution of the large extracellular M1P1 loop of TASK-3 with that from TASK-2, severely attenuated doxapram inhibition (8±4%, n=8) similar to that observed for WT TASK2 (7±5%, n=6).
Conclusion: Doxapram is an equally potent inhibitor of human TASK-1 and TASK-3 channels. This differs from rodent channels (3) and may help explain conflicting evidence from human and animal models. We have shown that an intracellular site on human TASK-3 channels is important for doxapram inhibition, however the large extracellular M1P1 loop may also have an important role in transducing the effect of the drug.
References: (1) Yost CS (2006). CNS Drug Review. 12:236-249. (2) Buckler KJ (2015). Pflugers Archiv. 467:1013-1023. (3) Cotten et al. (2006). Anesthesia & Analagesia. 102:779-785. (4) Chokshi RH et al. (2015). Mol Pharm. 88:926-934. (5) Clarke CE et al. (2008). J Biol Chem. 283:16985-16992.