Most cystic fibrosis (CF) patients produce a mutant form (delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR), which is not properly processed in normal cells but is active as a chloride channel in several experimental systems. We used a double homologous recombination (‘Hit and Run’) procedure to generate a mouse model for the delta F508 mutation. Targeted embryonic stem (ES) cells (Hit clones) were found; of these either 80 or 20% of the clones had lost the delta F508 mutation, depending on the distance between the linearization site in the targeting construct and the delta F508 mutation. Correctly targeted clones underwent a second selection step resulting in ES cell clones (Run clones) heterozygous for the delta F508 mutation with an efficiency of 2–7%. Chimeric mice were generated and offspring homozygous for the delta F508 mutation showed electrophysiological abnormalities in nasal epithelium, gallbladder and in the intestine, and histological abnormalities in the intestine, typical of CF. Our data suggest that the delta F508 mice have residual delta F508 CFTR activity which would explain the mild pathology of the delta F508 mice. The delta F508 mouse may provide a useful model for the study of the processing defect of delta F508 CFTR and for the development of novel therapeutic approaches based on circumvention of the processing block.

Compensatory mechanisms after genetic manipulations have been documented extensively for the nervous system. In many cases, these mechanisms involve genetic regulation at the transcription or expression level of existing isoforms. We report a novel mechanism by which single neurons compensate for changes in network connectivity by retuning their intrinsic electrical properties. We demonstrate this mechanism in the inferior olive, in which widespread electrical coupling is mediated by abundant gap junctions formed by connexin 36 (Cx36). It has been shown in various mammals that this electrical coupling supports the generation of subthreshold oscillations, but recent work revealed that rhythmic activity is sustained in knock-outs of Cx36. Thus, these results raise the question of whether the olivary oscillations in Cx36 knock-outs simply reflect the status of wild-type neurons without gap junctions or the outcome of compensatory mechanisms. Here, we demonstrate that the absence of Cx36 results in thicker dendrites with gap-junction-like structures with an abnormally wide interneuronal gap that prevents electrotonic coupling. The mutant olivary neurons show unusual voltage-dependent oscillations and an increased excitability that is attributable to a combined decrease in leak conductance and an increase in voltagedependent calcium conductance. Using dynamic-clamp techniques, we demonstrated that these changes are sufficient to transform a wild-type neuron into a knock-out-like neuron. We conclude that the absence of Cx36 in the inferior olive is not compensated by the formation of other gap-junction channels but instead by changes in the cytological and electroresponsive properties of its neurons, such that the capability to produce rhythmic activity is maintained.

The most prevalent mutation ( ∆ F508) in cystic fibrosis patients inhibits maturation and transfer to the plasma membrane of the mutant cystic fibrosis transmembrane conductance regulator (CFTR). We have analyzed the properties of a ⌬ F508 CFTR mouse model, which we described recently. We show that the mRNA levels of mutant CFTR are normal in all tissues examined. Therefore the reduced mRNA levels reported in two similar models may be related to their intronic transcription units. Maturation of mutant CFTR was greatly reduced in freshly excised oviduct, compared with normal. Accumulation of mutant CFTR antigen in the apical region of jejunum crypt enterocytes was not observed, in contrast to normal mice. In cultured gallbladder epithelial cells from ⌬ F508 mice, CFTR chloride channel activity could be detected at only two percent of the normal frequency. However, in mutant cells that were grown at reduced temperature the channel frequency increased to over sixteen percent of the normal level at that temperature. The biophysical characteristics of the mutant channel were not significantly different from normal. In homozygous ⌬ F508 mice we did not observe a significant effect of genetic background on the level of residual chloride channel activity, as determined by the size of the forskolin response in Ussing chamber experiments. Our data show that like its human homologue, mouse ⌬ F508-CFTR is a temperature sensitive processing mutant. The ⌬ F508 mouse is therefore a valid in vivo model of human ⌬ F508-CFTR. It may help us to elucidate the processing pathways of complex membrane proteins. Moreover, it may facilitate the discovery of new approaches towards therapy of cystic fibrosis. ( J. Clin. Invest. 1996. 98:1304-1312.)

Cysti c fi br osi s (CF), the most common sever e autosomal r ecessi ve tr ai t among Caucasi ans, i s caused by mol ecul ar l esi ons i n the cysti c fi br osi s tr ansmembr ane conductance r egul ator gene (CFTR). The cour se of the mul ti -or gan di sease CF i s hi ghl y var i abl e, suggesti ng the i nfl uence of envi r onmental factor s and/or modul ati ng genes other than CFTR on the di sease phenotype. To evaluate the cause of CF disease variability, the European CF Twin and Sibling Study col l ected data on tw o cl i ni cal par ameter s most sensi ti ve for the cour se and pr ognosi s of CF, i e w ei ght pr edi cted for hei ght (w fh)% (r epr esentati ve for the nutr i ti onal status) and FEVPer c (r epr esentati ve for the pul monar y status) for a cohor t of 277 si bl i ng pai r s, 12 pai r s of di zygous tw i ns and 29 pai r s of monozygous tw i ns. Of these 318 CF tw i n and si b pai r s, 114 w er e r epor ted to be homozygous for the most fr equent CF di sease-causi ng l esi on, ∆F508. I ntr a-pai r di scor dance w as assessed by the i ntr a-pai r di ffer ences w i th w fh% and FEVPer c and by DELTA , a composi te par ameter defi ned by l i near combi nati on of w fh% and FEVPer c i n or der to descr i be di scor dance w i th r espect to the overall disease severity. Monozygous twins had a significantly lower DELTA than dizygous twins (P = 0.05) i ndi cati ng that CF di sease sever i ty i s modul ated by an i nher i ted component i n addi ti on to the CFTR gene i tsel f. Extr eme phenotypes ar e consi der ed to be mor e i nfor mati ve for the anal ysi s of any quanti tati ve tr ai t. Thus, w e ai med to quanti fy di sease sever i ty and i ntr a-pai r di scor dance i n or der to sel ect pai r s w i th the extr eme phenotypes DI S (di scor dant pati ent pai r s), CON + (concor dant and mi l dy affected pati ent pai r s) and CON -(concor dant and sever el y affected pati ent pairs). The algorithm reliably discriminated between pairs DIS, CON + and CON -among the cohor t of ∆F508 homozygotes. The sel ected pai r s fr om these categor i es demonstr ated non-over l appi ng pr oper ti es for w fh%, FEVPer c and the i ntr a-pai r di ffer ence of both par ameter s. Twin Research (2000) 3, 277-293. Keyw or ds: cysti c fi brosi s di sease severi ty, affected rel ati ve pai r, tw i n and si bl i ng study, extreme phenotypes, al gori thm based sel ecti on I ntr oducti on Cysti c fi brosi s (CF) i s know n as the most common severe autosomal recessi ve di sease w i thi n the Caucasi an popul ati on, exhi bi ti ng an i nci dence of 1 i n 2500 bi rths.1 The symptoms of the di sorder are caused by an i mpai red functi on of exocri ne gl ands i n many organs, but major mani festati ons i nvol ve the respi ratory and the gastroi ntesti nal tracts.1 The di sease i s caused by mutati ons i n both chromosomal copi es of the cysti c fi brosi s transmembrane conductance regul ator (CFTR) gene. 2 The course of CF i s hi ghl y vari abl e w hen compari ng unrel ated pati ents w i th i denti cal CFTR mutati on genotypes, 3,4 o...

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Previous studies have revealed an adenosine 3',5'-cyclic monophosphate (cAMP)-independent activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by the tyrosine kinase inhibitor genistein. To further explore its mechanism of action, we have reconstituted genistein activation of CFTR in excised inside-out membrane patches. In the presence or absence of ATP, genistein appeared unable to open silent CFTR Cl- channels. However, on CFTR prephosphorylation by cAMP-dependent protein kinase (cAK), genistein enhanced CFTR activity by twofold, resulting from a prolonged burst duration. Genistein could also hyperactivate partially phosphorylated CFTR in the absence of cAK and therefore is different from 5'-adenylylimidodiphosphate, which required fully phosphorylated CFTR. Phosphatase-resistant thiophosphorylation likewise primed the CFTR Cl- channel for hyperactivation by genistein in the absence of cAK. Replacement of ATP by GTP as a hydrolyzable nucleotide triphosphate for CFTR did not impair the ability of genistein to activate thiophosphorylated CFTR, despite the fact that GTP is a poor substrate for tyrosine kinases. These findings argue against a role of protein phosphatases or tyrosine kinases but suggest a more direct interaction of genistein with CFTR, possibly at the level of the second nucleotide-binding domain.

Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type I␣ cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl ؊ channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP-dependent protein kinase (cAK) on opening CFTR-Cl ؊ channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl ؊ channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATP␥S (adenosine 5-O-(thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKI␣, is a potential activator of chloride transport in CFTR-expressing cell types.

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