Chimeric constructs endow the human CFTR Cl(-)channel with the gating behavior of murine CFTR

Scott-Ward, T. S., Cai, Z. W., Dawson, E. S., Doherty, A., Da Paula, A. C., Davidson, H., Porteous, D. J., Wainwright, B. J., Amaral, M. D., Sheppard, D. N. and Boyd, A. C. (2007) Chimeric constructs endow the human CFTR Cl(-)channel with the gating behavior of murine CFTR. Proceedings of The National Academy of Sciences of The United States of America, 104 41: 16365-16370. doi:10.1073/pnas.0701562104


Author Scott-Ward, T. S.
Cai, Z. W.
Dawson, E. S.
Doherty, A.
Da Paula, A. C.
Davidson, H.
Porteous, D. J.
Wainwright, B. J.
Amaral, M. D.
Sheppard, D. N.
Boyd, A. C.
Title Chimeric constructs endow the human CFTR Cl(-)channel with the gating behavior of murine CFTR
Journal name Proceedings of The National Academy of Sciences of The United States of America   Check publisher's open access policy
ISSN 0027-8424
Publication date 2007-01-01
Sub-type Article (original research)
DOI 10.1073/pnas.0701562104
Open Access Status Not Open Access
Volume 104
Issue 41
Start page 16365
End page 16370
Total pages 6
Place of publication Washington
Publisher National Academy of Sciences
Collection year 2008
Language eng
Subject C1
270299 Genetics not elsewhere classified
780105 Biological sciences
Abstract The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel gated by ATP-driven nucleotide-binding domain (NBD) dimerization. Here we exploit species differences between human and murine CFTR to investigate CFTR channel gating. Using homologous recombination, we constructed human-murine CFTR (hmCFTR) chimeras with sequences from NBD1, NBD2, or the regulatory domain (RD) of human CFTR replaced by the equivalent regions of murine CFTR. The gating behavior of hmRD and human CFTR were indistinguishable, whereas hmNBD1 and hmNBD2 had subtle effects on channel gating, prolonging both burst duration and interburst interval. By contrast, hmNBD1+2, containing both NBDs of murine CFTR, reproduced the gating behavior of the subconductance state of murine CFTR, which has dramatically prolonged channel openings. The CFTR potentiator pyrophosphate (PPi) enhanced human, hmRD, and hmNBD1 CFTR Cl- currents, but not those of hmNBD2, hmNBD1+2, and murine CFTR. By analyzing the rate-equilibrium free-energy relationships of chimeric channels, we obtained snapshots of the conformation of the NBDs during ATIP-driven dimerization. Our data demonstrate that the conformation of NBD1 changes before that of NBD2 during channel opening. This finding suggests that NBD dimerization does not proceed by a symmetric tweezer-like motion, but instead in an asymmetric fashion led by NBD1. We conclude that the NBDs of murine CFTR determine the unique gating behavior of its subconductance state, whereas NBD2 controls channel potentiation by PPi.
Keyword Multidisciplinary Sciences
ATP-binding cassette transporter
chloride ion channel
cystic fibrosis
recombinational cloning
rate-equilibrium free-energy relationships
Transmembrane-conductance-regulator
Nucleotide-binding Domains
Adenylate Kinase-activity
Cystic-fibrosis
Cl-channels
Wild-type
Mutations
Mechanism
Cloning
Cells
Q-Index Code C1
Q-Index Status Confirmed Code

 
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Created: Tue, 19 Feb 2008, 01:21:37 EST