title: An electrostatic interaction at the tetrahelix bundle promotes phosphorylation-dependent cystic fibrosis transmembrane conductance regulator (CFTR) channel opening.
abstract: The CFTR channel is an essential mediator of electrolyte transport across epithelial tissues. CFTR opening is promoted by ATP binding and dimerization of its two nucleotide binding domains (NBDs). Phosphorylation of its R domain (e.g. by PKA) is also required for channel activity. The CFTR structure is unsolved but homology models of the CFTR closed and open states have been produced based on the crystal structures of evolutionarily related ABC transporters. These models predict the formation of a tetrahelix bundle of intracellular loops (ICLs) during channel opening. Here we provide evidence that residues E267 in ICL2 and K1060 in ICL4 electrostatically interact at the interface of this predicted bundle to promote CFTR opening. Mutations or a thiol modifier that introduced like charges at these two positions substantially inhibited ATP-dependent channel opening. ATP-dependent activity was rescued by introducing a second site gain of function (GOF) mutation that was previously shown to promote ATP-dependent and ATP-independent opening (K978C). Conversely, the ATP-independent activity of the K978C GOF mutant was inhibited by charge- reversal mutations at positions 267 or 1060 either in the presence or absence of NBD2. The latter result indicates that this electrostatic interaction also promotes unliganded channel opening in the absence of ATP binding and NBD dimerization. Charge-reversal mutations at either position markedly reduced the PKA sensitivity of channel activation implying strong allosteric coupling between bundle formation and R domain phosphorylation. These findings support important roles of the tetrahelix bundle and the E267-K1060 electrostatic interaction in phosphorylation-dependent CFTR gating.
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center and Departments of Cell, Developmental, and Integrative Biology, and University of Alabama at Birmingham, Birmingham, Alabama 35294-0005. Electronic address: weiwang@uab.edu.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center and Departments of Cell, Developmental, and Integrative Biology, and University of Alabama at Birmingham, Birmingham, Alabama 35294-0005.
authors.forename: Bryan C
authors.initials: BC
authors.lastname: Roessler
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center and Departments of Cell, Developmental, and Integrative Biology, and University of Alabama at Birmingham, Birmingham, Alabama 35294-0005; Departments of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005. Electronic address: klkirk@uab.edu.
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Kirk
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008239:Lysine / Q000235:genetics; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D008239:Lysine
keywords: ABC Transporter; Allosteric Regulation; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR); Ion Channel; Phosphorylation