title: Mutations at the signature sequence of CFTR create a Cd(2+)-gated chloride channel.
abstract: The canonical sequence LSGGQ, also known as the signature sequence, defines the adenosine triphosphate (ATP)-binding cassette transporter superfamily. Crystallographic studies reveal that the signature sequence, together with the Walker A and Walker B motifs, forms the ATP-binding pocket upon dimerization of the two nucleotide-binding domains (NBDs) in a head-to-tail configuration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G551D) in cystic fibrosis transmembrane conductance regulator (CFTR) results in a severe phenotype of cystic fibrosis. We previously showed that the G551D mutation completely eliminates ATP-dependent gating of the CFTR chloride channel. Here, we report that micromolar [Cd(2+)] can dramatically increase the activity of G551D-CFTR in the absence of ATP. This effect of Cd(2+) is not seen in wild-type channels or in G551A. Pretreatment of G551D-CFTR with the cysteine modification reagent 2-aminoethyl methane thiosulfonate hydrobromide protects the channel from Cd(2+) activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd(2+). The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR's NBD1, show robust response to Cd(2+). On the other hand, negligible effects of Cd(2+) were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd(2+) binding to the gate. Collectively, these results suggest that the effect of Cd(2+) is mediated by a metal bridge formation between yet to be identified cysteine residue(s) and the engineered aspartate or cysteine in the signature sequence. We propose that the signature sequence serves as a switch that transduces the signal of ligand binding to the channel gate.
authors.affiliation: Department of Medical Pharmacology and Physiology, University of Missouri-Columbia, Columbia, MO 65211, USA.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Wang
authors.forename: Silvia G
authors.initials: SG
authors.lastname: Bompadre
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Tzyh-Chang
authors.initials: TC
authors.lastname: Hwang
mesh_terms: D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D013329:Structure-Activity Relationship
chemical_list: D008024:Ligands; D002104:Cadmium; D019005:Cystic Fibrosis Transmembrane Conductance Regulator