title: Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.
abstract: ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.
authors.affiliation: From the Departments of Cell, Developmental, and Integrative Biology.
authors.forename: Shipeng
authors.initials: S
authors.lastname: Wei
authors.affiliation: From the Departments of Cell, Developmental, and Integrative Biology.
authors.forename: Bryan C
authors.initials: BC
authors.lastname: Roessler
authors.affiliation: From the Departments of Cell, Developmental, and Integrative Biology.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Chauvet
authors.affiliation: Genetics, and.
authors.forename: Jingyu
authors.initials: J
authors.lastname: Guo
authors.affiliation: Genetics, and.
authors.forename: John L
authors.initials: JL
authors.lastname: Hartman
authors.affiliation: From the Departments of Cell, Developmental, and Integrative Biology, Neurobiology and the Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham Alabama 35294-0005 klkirk@uab.edu.
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Kirk
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D000266:Adenylyl Imidodiphosphate; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D050505:Mutant Proteins; D011392:Proline; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C505032:CFTR protein, human; C097077:YOR1 protein, S cerevisiae
keywords: ABC Transporter; Allosteric Regulation; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR); Ion Channel; Molecular Dynamics; Multidrug Transporter