title: Mammalian osmolytes and S-nitrosoglutathione promote Delta F508 cystic fibrosis transmembrane conductance regulator (CFTR) protein maturation and function.
abstract: In cystic fibrosis, the absence of functional CFTR results in thick mucous secretions in the lung and intestines, as well as pancreatic deficiency. Although expressed at high levels in the kidney, mutations in CFTR result in little or no apparent kidney dysfunction. In an effort to understand this phenomenon, we analyzed Delta F508 CFTR maturation and function in kidney cells under conditions that are common to the kidney, namely osmotic stress. Kidney cells were grown in culture and adapted to 250 mM NaCl and 250 mM urea. High performance liquid chromatography analysis of lysates from kidney cells adapted to these conditions identified an increase in the cellular osmolytes glycerophosphorylcholine, myo-inositol, sorbitol, and taurine. In contrast to isoosmotic conditions, hyperosmotic stress led to the proper folding and processing of Delta F508 CFTR. Furthermore, three of the cellular osmolytes, when added individually to cells, proved effective in promoting the proper folding and processing of the Delta F508 CFTR protein in both epithelial and fibroblast cells. Whole-cell patch clamping of osmolyte-treated cells showed that Delta F508 CFTR had trafficked to the plasma membrane and was activated by forskolin. Encouraged by these findings, we looked at other features common to the kidney that may impact Delta F508 maturation and function. Interestingly, a small molecule, S-nitrosoglutathione, which is a substrate for gamma glutamyltranspeptidase, an abundant enzyme in the kidney, likewise promoted Delta F508 CFTR maturation and function. S-Nitrosoglutathione-corrected Delta F508 CFTR exhibited a shorter half-life as compared with wild type CFTR. These results demonstrate the feasibility of a small molecule approach as a therapeutic treatment in promoting Delta F508 CFTR maturation and function and suggest that an additional treatment may be required to stabilize Delta F508 CFTR protein once present at the plasma membrane. Finally, our observations may help to explain why Delta F508 homozygous patients do not present with kidney dysfunction.
authors.affiliation: Department of Surgery, Surgical Research Laboratory, University of California, San Francisco, California 94110, USA. mbh1@itsa.ucsf.edu
authors.forename: Marybeth
authors.initials: M
authors.lastname: Howard
authors.forename: Horst
authors.initials: H
authors.lastname: Fischer
authors.forename: Jeremie
authors.initials: J
authors.lastname: Roux
authors.forename: Bento C
authors.initials: BC
authors.lastname: Santos
authors.forename: Steven R
authors.initials: SR
authors.lastname: Gullans
authors.forename: Paul H
authors.initials: PH
authors.lastname: Yancey
authors.forename: William J
authors.initials: WJ
authors.lastname: Welch
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001622:Betaine / Q000494:pharmacology; D002478:Cells, Cultured; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D007294:Inositol / Q000494:pharmacology; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000502:physiology; D008322:Mammals; D051379:Mice; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D026422:S-Nitrosoglutathione / Q000494:pharmacology*; D017384:Sequence Deletion; D013012:Sorbitol / Q000494:pharmacology; D013314:Stress, Mechanical; D013654:Taurine / Q000494:pharmacology; D014162:Transfection
chemical_list: D020030:Nitric Oxide Donors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D013654:Taurine; D001622:Betaine; D007294:Inositol; D013012:Sorbitol; D026422:S-Nitrosoglutathione