title: Thermally unstable gating of the most common cystic fibrosis mutant channel (ΔF508): "rescue" by suppressor mutations in nucleotide binding domain 1 and by constitutive mutations in the cytosolic loops.
abstract: Most cystic fibrosis (CF) cases are caused by the ΔF508 mutation in the CF transmembrane conductance regulator (CFTR), which disrupts both the processing and gating of this chloride channel. The cell surface expression of ΔF508-CFTR can be "rescued" by culturing cells at 26-28 °C and treating cells with small molecule correctors or intragenic suppressor mutations. Here, we determined whether these various rescue protocols induce a ΔF508-CFTR conformation that is thermally stable in excised membrane patches. We also tested the impact of constitutive cytosolic loop mutations that increase ATP-independent channel activity (K978C and K190C/K978C) on ΔF508-CFTR function. Low temperature-rescued ΔF508-CFTR channels irreversibly inactivated with a time constant of 5-6 min when excised patches were warmed from 22 °C to 36.5 °C. A panel of CFTR correctors and potentiators that increased ΔF508-CFTR maturation or channel activity failed to prevent this inactivation. Conversely, three suppressor mutations in the first nucleotide binding domain rescued ΔF508-CFTR maturation and stabilized channel activity at 36.5 °C. The constitutive loop mutations increased ATP-independent activity of low temperature-rescued ΔF508-CFTR but did not enhance protein maturation. Importantly, the ATP-independent activities of these ΔF508-CFTR constructs were stable at 36.5 °C, whereas their ATP-dependent activities were not. Single channel recordings of this thermally stable ATP-independent activity revealed dynamic gating and unitary currents of normal amplitudes. We conclude that: (i) ΔF508-CFTR gating is highly unstable at physiologic temperature; (ii) most rescue protocols do not prevent this thermal instability; and (iii) ATP-independent gating and the pore are spared from ΔF508-induced thermal instability, a finding that may inform alternative treatment strategies.
authors.affiliation: Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294. Electronic address: weiwang@uab.edu.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: George O
authors.initials: GO
authors.lastname: Okeyo
authors.affiliation: Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Binli
authors.initials: B
authors.lastname: Tao
authors.affiliation: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Jeong S
authors.initials: JS
authors.lastname: Hong
authors.affiliation: Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Kirk
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D001692:Biological Transport; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D003600:Cytosol / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D008957:Models, Genetic; D009154:Mutation; D009711:Nucleotides / Q000235:genetics; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: D009711:Nucleotides; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate