CFtr-orcc regulatory interaction requires autocrine atp transport, release and signaling

E. M. Schwiebert, M. E. Egan, S. S. Allen, Garry R Cutting, William B Guggino

Research output: Contribution to journalArticle

Abstract

CFTR regulates ORCCs through an autocrine signaling mechanism that requires CFTRand cyclic AMP-dependent ATP transport and release. ATP signaling occurs through purinergic receptors expressed on the apical membrane of the airway epithelium to stimulate ORCCs {Schwiebert et al. Celt 81:1063, 1995}. Thus, CFTR functions as a cAMP-regulated Cl channel and as a cAMP-regulated ATP channel which supplies ATP as an agonist to stimulate ORCCs. Two questions arise from these studies: (1) Which molecular domains of CFTR conduct Cl ions? and (2) which domains of CFTR transport ATP? To address these questions, we expressed a panel of mutated or truncated CFTR constructs by injecting Xenopus oocytes with CFTR cRNA and by lipofection of IB3-1 CF airway epithelial cells with RSV promoter-driven CFTR cDNA. Expression of two Nterminal truncated constructs, A259-M265, which lacks predicted a-helices 1-4 of CFTR, and A259-M265V, which lacks a-helices 1-4 with methionine 265 mutated to a valine, revealed different phenotypes. A259-M265 could conduct Cl and transport ATP to stimulate ORCCs: A259-M265V failed to conduct Cl but retained ATP transport function. TMD-1 CFTR, which contains only the first transmembrane domain of CFTR, functioned as a Cl channel but failed to transport ATP. T-N-R CFTR, which contains TMD-1, NBDI and the R domain of CFTR, retained both functions. CFTR carrying two mild CF lung disease-causing Cl conduction mutations, R334W and R347P, failed to conduct Cl but could transport ATP. Taken together, these data suggest that TMD-1 of CFTR conducts CT. while NBD-1 of CFTR is essential for ATP transport, release and signaling. These results show that the complex multi-domain structure of CFTR supports its multiple chloride channel and conductance regulator roles in epithelia. (Support: NRSA HL 08832).

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number3
StatePublished - 1996

Fingerprint

epithelium
Adenosine Triphosphate
chloride channels
cyclic AMP
Xenopus
valine
respiratory tract diseases
agonists
methionine
epithelial cells
oocytes
promoter regions
ions
mutation
phenotype
Epithelium
Autocrine Communication
Purinergic Receptors
Complementary RNA
Pulmonary diseases

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

CFtr-orcc regulatory interaction requires autocrine atp transport, release and signaling. / Schwiebert, E. M.; Egan, M. E.; Allen, S. S.; Cutting, Garry R; Guggino, William B.

In: FASEB Journal, Vol. 10, No. 3, 1996.

Research output: Contribution to journalArticle

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abstract = "CFTR regulates ORCCs through an autocrine signaling mechanism that requires CFTRand cyclic AMP-dependent ATP transport and release. ATP signaling occurs through purinergic receptors expressed on the apical membrane of the airway epithelium to stimulate ORCCs {Schwiebert et al. Celt 81:1063, 1995}. Thus, CFTR functions as a cAMP-regulated Cl channel and as a cAMP-regulated ATP channel which supplies ATP as an agonist to stimulate ORCCs. Two questions arise from these studies: (1) Which molecular domains of CFTR conduct Cl ions? and (2) which domains of CFTR transport ATP? To address these questions, we expressed a panel of mutated or truncated CFTR constructs by injecting Xenopus oocytes with CFTR cRNA and by lipofection of IB3-1 CF airway epithelial cells with RSV promoter-driven CFTR cDNA. Expression of two Nterminal truncated constructs, A259-M265, which lacks predicted a-helices 1-4 of CFTR, and A259-M265V, which lacks a-helices 1-4 with methionine 265 mutated to a valine, revealed different phenotypes. A259-M265 could conduct Cl and transport ATP to stimulate ORCCs: A259-M265V failed to conduct Cl but retained ATP transport function. TMD-1 CFTR, which contains only the first transmembrane domain of CFTR, functioned as a Cl channel but failed to transport ATP. T-N-R CFTR, which contains TMD-1, NBDI and the R domain of CFTR, retained both functions. CFTR carrying two mild CF lung disease-causing Cl conduction mutations, R334W and R347P, failed to conduct Cl but could transport ATP. Taken together, these data suggest that TMD-1 of CFTR conducts CT. while NBD-1 of CFTR is essential for ATP transport, release and signaling. These results show that the complex multi-domain structure of CFTR supports its multiple chloride channel and conductance regulator roles in epithelia. (Support: NRSA HL 08832).",
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