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In cystic fibrosis (CF), the AF508-CFTR anterograde trafficking from the endoplasmic reticulum to the plasma membrane is inefficient. New strategies for increasing the delivery of [DELTA]F508-CFTR to the apical membranes are thus pathophysiologically relevant targets to study for CF treatment. Recent studies have demonstrated that PDZ-containing proteins play an essential role in determining polarized plasma membrane expression of ionic transporters. In the present study we have hypothesized that the PDZ-containing protein NHE-RF1, which binds to the carboxy terminus of CFTR, rescues AF508-CFTR expression in the apical membrane of epithelial cells. The plasmids encoding [DELTA]F508-CFTR and NHE-RF1 were intranuclearly injected in A549 or Madin-Darby canine kidney (MDCK) cells, and [DELTA]F508-CFFR channel activity was functionally assayed using SPQ fluorescent probe. Cells injected with [DELTA]F508-CFTR alone presented a low chloride channel activity, whereas its coexpression with NHE-RF1 significantly increased both the basal and forskolin-activated chloride conductances. This last effect was lost with [DELTA]F508-CFTR deleted of its 13 last amino acids or by injection of a specific NHE-RF1 antisense oligonucleotide, but not by NHE-RFI sense oligonucleotide. Immunocytochemical analysis performed in MDCK cells transiently transfected with [DELTA]F508-CFTR further revealed that NHE-RF1 specifically determined the apical plasma membrane expression of [DELTA]F508-CFTR but not that of a trafficking defective mutant potassium channel (KCNQ1). These data demonstrate that the modulation of the expression level of CFTR protein partners, like NHE-RF1, can rescue [DELTA]F508-CFTR activity. cystic fibrosis; [DELTA]F508 cystic fibrosis transmembrane conductance regulator; [Na.sup.+]/[H.sup.+] exchanger regulatory factor isoform 1; polarized expression; traffic doi:10.1152/ajplung.00445.2005

Bulteau, Laurence, Renaud Derand, Yvette Mettey, Thierry Metaye, M. Rachel Morris, Ceinwen M. McNeilly, Chiara Folli, Luis J. V. Galietta, Olga Zegarra-Moran, Malcolm M. C. Pereira, Chantal Jougla, Robert L. Dormer, Jean-Michel Vierfond, Michel Joffre, and Frederic Becq. Properties of CFTR activated by the xanthine derivative X-33 in human airway Calu-3 cells. Am J Physiol Cell Physiol 279: C1925-C 1937, 2000.--The pharmacological activation of the cystic fibrosis gene protein cystic fibrosis transmembrane conductance regulator (CFTR) was studied in human airway epithelial Calu-3 cells, which express a high level of CFTR protein as assessed by Western blot and in vitro phosphorylation. Immunolocalization shows that CFTR is located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel synthesized xanthine derivative 3,7-dimethyl-1-isobutylxanthine (X-33) is an activator of the CFTR channel in Calu-3 cells. Whole cell current activated by X-33 or IBMX is linear, inhibited by glibenclamide and diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene. Intracellular cAMP was not affected by X-33. An outwardly rectifying [Cl.sup.-] current was recorded in the absence of cAMP and X-33 stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use of short-circuit recordings, X-33 and IBMX were able to stimulate a large concentration-dependent CFTR transport that was blocked by glibenclamide but not by DIDS. Our results show that manipulating the chemical structure of xanthine derivatives offers an opportunity to identify further specific activators of CFTR in airway cells. cystic fibrosis transmembrane conductance regulator; chloride conductance; pharmacology

Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation. Am J Physiol Cell Physiol 281: C1657-C1666, 2001.--We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (E[C.sub.50] = 85 [micro]M) and activated CFTR in Calu-3 cells (E[C.sub.50] = 47 [micro]M). MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity. pharmacology; disease-causing mutation; cystic fibrosis; nucleotide binding domains; cystic fibrosis transmembrane conductance regulator