期刊
JOURNAL OF PHYSIOLOGY-LONDON
卷 539, 期 2, 页码 333-346出版社
WILEY
DOI: 10.1113/jphysiol.2001.013162
关键词
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资金
- NHLBI NIH HHS [R01 HL053445] Funding Source: Medline
- NIDDK NIH HHS [R01 DK055835] Funding Source: Medline
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R01HL053445] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [R01DK055835] Funding Source: NIH RePORTER
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 (K464A) using the patch clamp technique. K464A mutants have an ATP dependence (EC50 approximate to60 muM) and opening rate at 2.75 mm ATP (similar to2.1 s(-1)) similar to wild type (EC50 approximate to 97 muM; similar to 2.0 s(-1)). However, K464A's closing rate at 2.75 mM a ATP (similar to 3.6 s(-1)) is faster than that of wild type (similar to 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 (similar to 2- and similar to 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 (similar to 0.055 s(-1)) and closing (similar to 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 similar to 5-fold faster (similar to 0.029 s(-1)) than KI 250A but open with a similar rate (similar to 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.
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