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Mutation of Walker‐A lysine 464 in cystic fibrosis transmembrane conductance regulator reveals functional interaction between its nucleotide‐binding domains
Author(s) -
Powe Allan C.,
AlNakkash Layla,
Li Min,
Hwang TzyhChang
Publication year - 2002
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2001.013162
Subject(s) - cystic fibrosis transmembrane conductance regulator , chloride channel , mutant , nucleotide , gating , chemistry , walker motifs , biophysics , wild type , biochemistry , atp hydrolysis , biology , atpase , enzyme , gene
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel bears two nucleotide‐binding domains (NBD1 and NBD2) that control its ATP‐dependent gating. Exactly how these NBDs control gating is controversial. To address this issue, we examined channels with a Walker‐A lysine mutation in NBD1 (K464 A) using the patch clamp technique. K464A mutants have an ATP dependence (EC 50 ≈ 60 μ m ) and opening rate at 2.75 m m ATP (∼ 2.1 s −1 ) similar to wild type (EC 50 ≈ 97 μ m ; ∼ 2.0 s −1 ). However, K464A's closing rate at 2.75 m m ATP (∼ 3.6 s −1 ) is faster than that of wild type (∼ 2.1 s −1 ), suggesting involvement of NBD1 in nucleotide‐dependent closing. Delay of closing in wild type by adenylyl imidodiphosphate (AMP‐PNP), a non‐hydrolysable ATP analogue, is markedly diminished in K464A mutants due to reduction in AMP‐PNP's apparent on‐rate and acceleration of its apparent off‐rate (∼ 2‐ and ∼ 10‐fold, respectively). Since the delay of closing by AMP‐PNP is thought to occur via NBD2, K464A's effect on the NBD2 mutant K1250A was examined. In sharp contrast to K464A, K1250A single mutants exhibit reduced opening (∼ 0.055 s −1 ) and closing (∼ 0.006 s −1 ) rates at millimolar [ATP], suggesting a role for K1250 in both opening and closing. At millimolar [ATP], K464A‐K1250A double mutants close ∼ 5‐fold faster (∼ 0.029 s −1 ) than K1250A but open with a similar rate (∼ 0.059 s −1 ), indicating an effect of K464A on NBD2 function. In summary, our results reveal that both of CFTR's functionally asymmetric NBDs participate in nucleotide‐dependent closing, which provides important constraints for NBD‐mediated gating models.