HANRAHAN, J.W.* *; ZHU, T.; DAHAN, D.; FUNG, S.; MEREDITH, J.; PHILLIPS, J.E.: The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel

CFTR is a phosphorylation-regulated chloride channel and member of the ATP binding cassette (ABC) transporter superfamily of proteins. There have been no reports of CFTR homologues in invertebrate animals although ABC transporters are found in virtually all organisms from bacteria to humans. The hindgut of the desert locust Schistocerca gregaria expresses a cAMP-activated chloride conductance reminiscent of CFTR. We carried out preliminary RT-PCR studies using RNA from this tissue. Several primer sets failed to amplify CFTR-like products. A search of the Drosophila genome revealed many putative ABC transporters but none having the regulatory (R) domain diagnostic of CFTR. Thus CFTR may have appeared late in evolution compared to other ABC transporters and channels. CFTR channels rapidly deactivate in excised membrane patches suggesting they may be associated with a membrane-bound phosphatase. We tested that possibility using co-immunoprecipitation and crosslinking studies. A monoclonal anti-CFTR antibody co-precipitated PP2C (but not PP1, PP2A or PP2B) from cells stably expressing CFTR. Conversely, a polyclonal anti-PP2C antibody co-precipitated CFTR from membrane extracts. When cell lysates were exposed to the bifunctional crosslinking reagent dithiobis [sulfosuccinimidyl propionate] (DTSSP), histidine-tagged CFTR (CFTR_His10) and PP2C became crosslinked into high molecular weight complexes that could be isolated by chromatography on Ni²⁺-NTA agarose. PP1, PP2A and PP2B were not co-purified. These results suggest CFTR and PP2C exist in a stable complex that facilitates regulation of the channel. SDS-PAGE and mass spectrometry are being used to identify the PP2C isoform.