title: A single amino acid substitution in CFTR converts ATP to an inhibitory ligand.
abstract: Cystic fibrosis (CF), one of the most common lethal genetic diseases, is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel that, when phosphorylated, is gated by ATP. The third most common pathogenic mutation, a glycine-to-aspartate mutation at position 551 or G551D, shows a significantly decreased open probability (Po) caused by failure of the mutant channel to respond to ATP. Recently, a CFTR-targeted drug, VX-770 (Ivacaftor), which potentiates G551D-CFTR function in vitro by boosting its Po, has been approved by the FDA to treat CF patients carrying this mutation. Here, we show that, in the presence of VX-770, G551D-CFTR becomes responsive to ATP, albeit with an unusual time course. In marked contrast to wild-type channels, which are stimulated by ATP, sudden removal of ATP in excised inside-out patches elicits an initial increase in macroscopic G551D-CFTR current followed by a slow decrease. Furthermore, decreasing [ATP] from 2 mM to 20 µM resulted in a paradoxical increase in G551D-CFTR current. These results suggest that the two ATP-binding sites in the G551D mutant mediate opposite effects on channel gating. We introduced mutations that specifically alter ATP-binding affinity in either nucleotide-binding domain (NBD1 or NBD2) into the G551D background and determined that this disease-associated mutation converts site 2, formed by the head subdomain of NBD2 and the tail subdomain of NBD1, into an inhibitory site, whereas site 1 remains stimulatory. G551E, but not G551K or G551S, exhibits a similar phenotype, indicating that electrostatic repulsion between the negatively charged side chain of aspartate and the γ-phosphate of ATP accounts for the observed mutational effects. Understanding the molecular mechanism of this gating defect lays a foundation for rational drug design for the treatment of CF.
authors.affiliation: Department of Medical Pharmacology and Physiology, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211 Department of Medical Pharmacology and Physiology, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211.
authors.forename: Wen-Ying
authors.initials: WY
authors.lastname: Lin
authors.affiliation: Department of Medical Pharmacology and Physiology, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211 Physician-Scientist Program, National Yang-Ming University, Taipei, 112 Taiwan.
authors.forename: Kang-Yang
authors.initials: KY
authors.lastname: Jih
authors.affiliation: Department of Medical Pharmacology and Physiology, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211 Department of Medical Pharmacology and Physiology, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211 hwangt@health.missouri.edu.
authors.forename: Tzyh-Chang
authors.initials: TC
authors.lastname: Hwang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology*; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation / Q000502:physiology; D018408:Patch-Clamp Techniques; D011487:Protein Conformation
chemical_list: D018076:DNA, Complementary; D008024:Ligands; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases