CFTR chloride channel regulation by an interdomain interaction

Science

Volumn 286, Issue 5439, 1999, Pages 544-548

  Naren, Anjaparavanda P ^a   Cormet Boyaka, Estelle ^a   Fu, Jian ^a   Villain, Matteo ^a   Blalock, J Edwin ^a   Quick, Michael W ^a,b   Kirk, Kevin L ^a,b  

^a UNIVERSITY OF ALABAMA AT BIRMINGHAM   (United States)

^b University of Alabama at Birmingham   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CHLORIDE CHANNEL; CYCLIC AMP DEPENDENT PROTEIN KINASE; TRANSMEMBRANE CONDUCTANCE REGULATOR;

AMINO TERMINAL SEQUENCE; ARTICLE; CHANNEL GATING; PRIORITY JOURNAL; PROTEIN DOMAIN; PROTEIN PHOSPHORYLATION;

ADENOSINE TRIPHOSPHATE; AMINO ACID SEQUENCE; AMINO ACID SUBSTITUTION; ANIMALS; COS CELLS; CYCLIC AMP; CYCLIC AMP-DEPENDENT PROTEIN KINASES; CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR; DNA MUTATIONAL ANALYSIS; HUMANS; ION CHANNEL GATING; MOLECULAR SEQUENCE DATA; MUTATION; OOCYTES; PATCH-CLAMP TECHNIQUES; PHOSPHORYLATION; PROTEIN STRUCTURE, SECONDARY; RECOMBINANT FUSION PROTEINS; XENOPUS;

EID: 0033569511     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5439.544     Document Type: Article

References (27)

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12. Cystic Fibrosis Genetic Analysis Consortium (www. genet.sickkids.on.ca/cftr).
13. All point mutations were introduced into a 1-kb fragment of the CFTR coding region by the Quick-change site-directed mutagenesis kit (Stratagene). This fragment was sequenced completely to verify each mutation and then ligated into pCDNA3 vector (Invitrogen) containing the rest of the CFTR coding region.
14. A. P. Naren, E. Cormet-Boyaka, K. L. Kirk, unpublished data.
15. A. P. Naren, M. Villain, D. Muccio, K. L. Kirk, unpublished data.
16. The triple and quadruple N-Tail mutants are not completely inactive; for example, cAMP-activated currents can be detected when higher cRNA amounts for these mutants are injected. Indeed, because of the nonlinearity of the oocyte expression system, the currents mediated by these mutants can approach (but not reach) wild-type levels at very high cRNA amounts (such as 50 ng).
17. J. Fu and K. L. Kirk, unpublished data.
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19. M. W. Quick and K. L. Kirk, unpublished data.
20. Recombinant NBD1 (amino acids 433 to 584) bound weakly to GST-N-Tail, but this binding was not inhibited by the N-Tail mutations that inhibit CFTR channel activity.
21. A. P. Naren and K. L. Kirk, unpublished data.
22. M. C. Winter and M. J. Welsh, Nature 387, 294 (1997).
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24. Assays were done as described (9, 10). Briefly, soluble GST-N-Tail peptide was added to a lysate of COS-7 cells [0.2% Triton X-100 in phosphate-buffered saline (PBS)] transiently expressing recombinant CFTR (or various R domain constructs) and mixed for 1 hour at room temperature. Bound proteins were precipitated with excess glutathione agarose, washed extensively in 0.2% Triton X-100 in PBS, and analyzed for CFTR or R domain by immunoblotting with monoclonal antibodies to the COOH-terminus or R domain (Genzyme). CFTR immunoprecipitations were done on portions of the same lysates (9, 10).
25. 2-terminus with fluorenyl methoxycarbonyl-amino caproic acid as spacer. Peptide binding (biotin P30-63) was assessed by mixing 1.25 μM peptide and 2.5 μM soluble GST-R domain peptide in PBS for 1 hour at 22°C. A 100-fold molar excess of unbiotinylated peptide was used for competition experiments. The complex was precipitated with excess glutathione agarose and washed with PBS. Streptavidin-horseradish peroxidase (HRP, 1 μM) was added in PBS and incubated for 20 min. The beads were washed extensively in PBS plus 0.2% Triton X-100 and assayed for HRP activity with 2,2′-azinobis(3-ethylbenzothiozoline)-6 sulfonic acid (ABTS) according to manufacturer's instructions (Pierce, St. Louis, MO).
26. 2, 1 mM EGTA, and 10 mM Hepes (pH to 7.4 with HCl) supplemented with 1.5 mM Mg-ATP and PKA catalytic subunit (80 U/ml, Promega). Records of multichannel patches (holding potential -80 mV) were digitized using an Axopatch 200B amplifier, filtered at 200 Hz, and analyzed using PCLAMP 5.0 software. The open probabilities of the N-Tail mutants should be considered to be maximal estimates given the very brief open times of these mutants, which leads to underestimating channel number. Open-channel burst durations were estimated with the cycle time method and a minimal interburst duration of 20 ms (8). The N-Tail mutants also exhibited reduced open-channel burst durations relative to wild-type CFTR when analyzed at shorter minimal interburst durations such as 10 ms; however, this gave an underestimate of the true open-channel burst duration. The single-channel properties of the ΔR CFTR constructs were analyzed in membrane patches excised from transfected HeLa cells as described (22) with a minimal interburst duration of 20 ms. All patch clamp experiments were performed at 20° to 22°C.
27. We thank M. Welsh for providing the original ΔR-S660A CFTR construct. Supported by NIH grants DK51868 and DK50830 (K.L.K.), DA10509 (M.W.Q.), and MH52527 (J.E.B.).