TY - JOUR T1 - Breathing Stimulant Compounds Inhibit TASK-3 Potassium Channel Function Likely by Binding at a Common Site in the Channel Pore JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.115.100107 SP - mol.115.100107 AU - Rikki H. Chokshi AU - Aaron T. Larsen AU - Brijesh Bhayana AU - Joseph F. Cotten Y1 - 2015/01/01 UR - http://molpharm.aspetjournals.org/content/early/2015/08/12/mol.115.100107.abstract N2 - PKTHPP, A1899, and doxapram are compounds that inhibit TASK-1 (KCNK3) and TASK-3 (KCNK9) tandem pore (K2P) potassium channel function and that stimulate breathing. To better understand the molecular mechanism(s) of action of these drugs, we undertook studies to identify amino acid residues in the TASK-3 protein that mediate this inhibition. HYPOTHESIS: Guided by homology modeling and molecular docking, we hypothesized PKTHPP and A1899 bind in the TASK-3 intracellular pore. METHODS: To test our hypothesis, we mutated each residue in or near the predicted PKTHPP and A1899 binding site (residues 118 to 128 and 228 to 248), individually, to a negatively charged aspartate. We quantified each mutation's effect on TASK-3 potassium channel concentration-response to PKTHPP. Studies were conducted on TASK-3 transiently expressed in a Fischer rat thyroid epithelial monolayers; channel function was measured in an Ussing chamber. RESULTS: TASK-3 pore mutations at residues-122 (L122D, E, or K) and -236 (G236D) caused the IC50 of PKTHPP to increase more than 1000-fold. TASK-3 mutants L122D, G236D, L239D, and V242D were resistant to block by PKTHPP, A1899, and doxapram. CONCLUSIONS: Our data are consistent with a model in which breathing stimulant compounds PKTHPP, A1899, and doxapram inhibit TASK-3 function by binding at a common site within the channel intracellular pore region, although binding outside the channel pore can not yet be excluded. ER -