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, defi nes 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 confi guration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G551D) in cystic fi brosis transmembrane conductance regulator (CFTR) results in a severe phenotype of cystic fi brosis. 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 modifi cation 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 specifi c 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 identifi ed 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. © 2009 Wang et al. © 2009 Wang et al.