Mutations in the TASK‐3 Potassium Channel Pore Modify Its Inhibition by Breathing Stimulant Compounds

Abstract

BACKGROUND Drugs that inhibit TASK-1 and TASK-3 potassium channel function (e.g., PKTHPP, A1899, and doxapram) cause stimulation of breathing in rodents. To better understand the mechanism(s) of action of these drugs, we undertook studies to identify residues in TASK-3 that mediate this inhibition. HYPOTHESIS: Guided by in silico drug docking, we hypothesized PKTHPP and A1899 bind in the TASK-3 intracellular pore. METHODS: To test our hypothesis, we mutated residues in or near the predicted PKTHPP and A1899 binding site (residues 120 to 128). We quantified each mutation's effect on the TASK-3 concentration-response to PKTHPP. Studies were conducted on TASK-3 transiently expressed in a Fischer rat thyroid cell line; channel function was measured by Ussing chamber. RESULTS: One mutant, TASK-3 L122D, was markedly resistant to block by PKTHPP, A1899, and doxapram (Table 1). Other pore mutations had more modest effects. Three residues when mutated (L120D, M124D, L128D) were without function. Conclusions Our data are consistent with a model in which breathing stimulant drugs PKTHPP, A1899, and doxapram inhibit TASK-3 function by binding within the channel intracellular pore. FUNDING: Massachusetts General Hospital Department of Anesthesia, Critical Care & Pain Medicine; NIH/HL117871; NSF/CHE0809413; Simons Collaboration on the Origins of Life. IC50 Wild-type T121D L122D V123D M125D Q126D S127D PKTHPP (nM) 10 (9-11) 19 (15-24)* >10,000t 5 (4-7)* 31 (21-48)* 160 (96-265)* 575 (522-635)* A1899 (nM) 18 (15-22) nd >10,000t nd nd nd nd Doxapram (uM) 16 (11-21 ) nd 1,300 (1,100-1,700)* nd nd nd nd