Breathing Stimulant Compounds Inhibit TASK-3 Potassium Channel Function Likely by Binding at a Common Site in the Channel Pore.
Compounds PKTHPP (1-{1-[6-(biphenyl-4-ylcarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]-pyrimidin-4-yl]piperidin-4-yl}propan-1-one), A1899 (2''-[(4-methoxybenzoylamino)methyl]biphenyl-2-carboxylic acid 2,4-difluorobenzylamide), and doxapram inhibit TASK-1 (KCNK3) and TASK-3 (KCNK9) tandem pore (K2P) potassium channel function and 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. Guided by homology modeling and molecular docking, we hypothesized that PKTHPP and A1899 bind in the TASK-3 intracellular pore. To test our hypothesis, we mutated each residue in or near the predicted PKTHPP and A1899 binding site (residues 118-128 and 228-248), 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 Fischer rat thyroid epithelial monolayers; channel function was measured in an Ussing chamber. 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. 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 cannot yet be excluded.
Department of Anesthesia, Critical Care, and Pain Medicine (R.H.C., J.F.C.), Center for Computational and Integrative Biology, and Department of Molecular Biology (A.T.L.), and Department of Dermatology (B.B.), Massachusetts General Hospital, Boston, Massachusetts.
Rikki H
RH
Chokshi
Department of Anesthesia, Critical Care, and Pain Medicine (R.H.C., J.F.C.), Center for Computational and Integrative Biology, and Department of Molecular Biology (A.T.L.), and Department of Dermatology (B.B.), Massachusetts General Hospital, Boston, Massachusetts.
Aaron T
AT
Larsen
Department of Anesthesia, Critical Care, and Pain Medicine (R.H.C., J.F.C.), Center for Computational and Integrative Biology, and Department of Molecular Biology (A.T.L.), and Department of Dermatology (B.B.), Massachusetts General Hospital, Boston, Massachusetts.
Brijesh
B
Bhayana
Department of Anesthesia, Critical Care, and Pain Medicine (R.H.C., J.F.C.), Center for Computational and Integrative Biology, and Department of Molecular Biology (A.T.L.), and Department of Dermatology (B.B.), Massachusetts General Hospital, Boston, Massachusetts jcotten@mgh.harvard.edu.
Joseph F
JF
Cotten
D000595:Amino Acid Sequence; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D044482:Benzeneacetamides / Q000494:pharmacology; D001665:Binding Sites; D002478:Cells, Cultured; D004315:Doxapram / Q000494:pharmacology; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D024683:Potassium Channels, Tandem Pore Domain / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000502:physiology; D051381:Rats; D011916:Rats, Inbred F344; D019141:Respiratory System Agents / Q000378:metabolism / Q000494:pharmacology*; D013329:Structure-Activity Relationship
C558924:A 1899; D001549:Benzamides; D044482:Benzeneacetamides; C498655:KCNK9 protein, human; D024683:Potassium Channels, Tandem Pore Domain; D019141:Respiratory System Agents; D004315:Doxapram