Cell Biology And Molecular Pharmacology Of Pig Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) And â–³F508-pCFTR

The cystic fibrosis transmembrane conductance regulator(CFTR) is a cAMP-activated Cl^- channel expressed in secretory and absorptive epithelia in the airways,pancreas, gastrointestinal tract,testis,sweat gland,salivary gland and other tissues.It is involved in transport of electrolyte,fluid and regulation of other membrane channel proteins.Mutations in CFTR cause cystic fibrosis(CF),the most common lethal autosomal recessive genetic disease involving multisystem disorders in the Caucasian population.Deletion of the codon encoding the phenylalanine residue at position 508(ΔF508) in CFTR is the most common mutation of CF and appears in one allele of～90%CF patients.The disease is characterized by recurrent lung infections with opportunistic pathogens,which results in progressive and irreversible loss of lung function.The airway disease is currently the source of most CF morbidity and mortality.A major impediment to understand CF pathogenesis and to develop new treatment is the limitation of current animal models.Although many lines of CF mice have been generated by targeting the mouse CFTR gene after cloning of CFTR,these CF mouse models manifest some intestinal phenotypes of CF patients.They have failed to develop the spontaneous lung infections seen in patients with CF.This failure may be due to differences in airway cell biology,the abundance of submucosal glands in the airway,and the alternative expression or activation of pathways that control non-CFTR Cl^- channels in airways.This has prompted researchers in the CF field to attempt to identify or generate models for human CF in other animals.The pig represents another potentially useful alternative species for generating a CF model due to the similarities between its lung anatomical and physiological features and human lung's.Further more,the longevity of pigs also offers opportunities for investigating the pathogenesis of lung disease,the long-term therapeutic efficacy of treatments and adverse effects of interventions that might only become apparent with time. Recent advances were reported in generating transgenic pig models withΔF508 and null mutations of CFTR.However,the null mutation of CF pig maybe die from intestinal obstruction at early age like the CF mice and this presents a technical challenge when trying to use these pigs for experimentation.In another way,a recent study reported by Osedgaad et al indicated partial cellular processing and significant plasma membrane targeting of porcineΔF508-CFTR,raising cautions on the phenotypic consequences of theΔF508 pig model.Then there is considerable interest in development pig CF models by using pig CFTR specific, high-affinity inhibitors.However,the human CFTR inhibitor CFTR_inh-172 produced a significantly lower level of inhibition of pig CFTR and our previous work failed to create pig CF models by in vivo administration of CFTR_inh-172.Another specific human CFTR inhibitor GlyH101 is also a potent inhibitor of pig CFTR,but its organ distribution in animals is not clear.Therefore we are not sure if GlyH101 is suitable for developing CF pig models.What we need is a high-affinity,specific inhibitor of pig CFTR to create CF lung phenotypes in pig.The purpose of this study was to characterize the biochemical and electrophysiological features of pigΔF508-CFTR and to discover new pig CFTR inhibitors by high throughput screening.Firstly,we cloned the cDNA sequence encoding full-length porcine CFTR(pCFTR) by RT-PCR and subcloned it to expression vector pcDNA3.1Zeo to form recombinant plasmid pcDNA3.1Zeo/pCFTR.TheΔF508 mutation of pig CFTR was generated in pcDNA3.1ZeopCFTR by site-directed mutagenesis to form pcDNA3.1ZeoΔF508-pCFTR using a PCR-based strategy.Then we established stably cotransfected FRT cell lines coexpressing the wildtype or mutant pig CFTRs and iodide-sensitive green fluorescent analog EYFP-H148Q-I152L as cell-based assay models for functional analysis and high-throughput screening.Western blot analysis and immunofluorescence of Cos7 and FRT cell by using the generated pig CFTR polyclonal antibody against the NBD2 domain revealed predominant plasma membrane targeting(77.8%) ofΔF508-pCFTR protein.Functinal measurements by fluorometric assay,short circuit current assay and whole-cell patch-clamp assay indicated thatΔF508-pCFTR produced a cAMP-activated Cl^- current.However,the low sensitivity ofΔF508-pCFTR to cAMP agonist forskolin and high sensitivity to CFTR inhibitor suggested reduced channel activity ofΔF508-pCFTR.It was estimated that plasma membrane targeting of 10%ΔF508-CFTR protein could avoid the CF phenotype in human subjects.The mild processing defect of porcineΔF508-CFTR arouses suspicion that the ongoing transgenicΔF508-CFTR pig model may not develop desired CF phenotypes.Secondly,we analyze the distribution of GlyH101 in mouse tissues and found low distribution and rapid clearance of GlyH101 in mouse lung after intraperitoneal injection, which suggested that GlyH101 is not suitable for developing pig CF models.Lastly,we performed high-throughput screening of 100,000 synthetic small molecules to identify new chemical scaffolds with pig CFTR inhibitory activity.We obtained 3 new pig CFTR inhibitors PI1,PI2 and PI3.The most potent CFTR inhibitor was PI1 with IC50 value about 14μM.The inhibition was rapid,reversible,and nontoxic.Further more,it could inhibit mouse intestinal fluid secretion induced by cholera toxin.The present study confirmed the species difference in cellular processing ofΔF508-CFTR.However,our results indicated rather mild processing defect ofΔF508-pCFTR. It was estimated that plasma membrane targeting of 10%ΔF508-CFTR protein could avoid the CF phenotype in human subjects.The mild processing defect of porcineΔF508-CFTR arouses suspicion that the ongoing transgenicΔF508-CFTR pig model may not develop desired CF phenotypes. We identified 3 new pig CFTR inhibitors from 100,000 synthetic small molecules. However,the poor affinity of these inhibitors potentially precluded their usefulness in generating CF phenotypes in pig.Future direction to discover potent pig CFTR inhibitors will involve screening of a natural compounds library recently established in our laboratory to identify high-affinity pig CFTR inhibitors instead of small molecules.