title: Chimeric constructs endow the human CFTR Cl- channel with the gating behavior of murine CFTR.
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).
authors.affiliation: Department of Physiology and Pharmacology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, United Kingdom.
authors.forename: Toby S
authors.initials: TS
authors.lastname: Scott-Ward
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Cai
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Dawson
authors.forename: Ann
authors.initials: A
authors.lastname: Doherty
authors.forename: Ana Carina
authors.initials: AC
authors.lastname: Da Paula
authors.forename: Heather
authors.initials: H
authors.lastname: Davidson
authors.forename: David J
authors.initials: DJ
authors.lastname: Porteous
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Wainwright
authors.forename: Margarida D
authors.initials: MD
authors.lastname: Amaral
authors.forename: David N
authors.initials: DN
authors.lastname: Sheppard
authors.forename: A Christopher
authors.initials: AC
authors.lastname: Boyd
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D003550:Cystic Fibrosis / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D011756:Diphosphates / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D007700:Kinetics; D051379:Mice; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity
chemical_list: C505032:CFTR protein, human; D011756:Diphosphates; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate