1. Chimeric constructs endow the human CFTR Cl- channel with the gating behavior of murine CFTR.
- Author
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Scott-Ward TS, Cai Z, Dawson ES, Doherty A, Da Paula AC, Davidson H, Porteous DJ, Wainwright BJ, Amaral MD, Sheppard DN, and Boyd AC
- Subjects
- Adenosine Triphosphate pharmacology, Animals, Binding Sites, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator antagonists & inhibitors, Cystic Fibrosis Transmembrane Conductance Regulator chemistry, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Dimerization, Diphosphates pharmacology, Humans, In Vitro Techniques, Ion Channel Gating, Kinetics, Mice, Protein Structure, Tertiary, Recombinant Fusion Proteins antagonists & inhibitors, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Species Specificity, Cystic Fibrosis Transmembrane Conductance Regulator metabolism
- 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 (PP(i)) 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 ATP-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 PP(i).
- Published
- 2007
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