期刊
JOURNAL OF GENERAL PHYSIOLOGY
卷 133, 期 1, 页码 69-77出版社
ROCKEFELLER UNIV PRESS
DOI: 10.1085/jgp.200810049
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资金
- Cystic Fibrosis Foundation [BOMPAD06G0]
- National Institutes of Health [K01 DK075408]
- National Center for Research Resources, National Institutes of Health [C06 RR-016489-01]
- [NIHR01DK55835]
- [NIHR01HL53455]
- [CFFT CLARKE06XX0]
- NATIONAL CENTER FOR RESEARCH RESOURCES [K23RR016489] Funding Source: NIH RePORTER
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R01HL053455] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [R01DK055835, K01DK075408] Funding Source: NIH RePORTER
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 [Cd2+] can dramatically increase the activity of G551D-CFTR in the absence of ATP. This effect of Cd2+ 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 Cd2+ activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd2+. The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR's NBD1, show robust response to Cd2+. On the other hand, negligible effects of Cd2+ were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd2+ binding to the gate. Collectively, these results suggest that the effect of Cd2+ 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.
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