A Monomer Is the Minimum Functional Unit Required for Channel and ATPase Activity of the Cystic Fibrosis Transmembrane Conductance Regulator†

Biochemistry ◽  
2001 ◽  
Vol 40 (35) ◽  
pp. 10700-10706 ◽  
Author(s):  
Mohabir Ramjeesingh ◽  
Canhui Li ◽  
Ilana Kogan ◽  
Yanchun Wang ◽  
Ling-Jun Huan ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Functional Unit ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

scholarly journals Perturbation of the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Inhibits Its ATPase Activity

2000 ◽  
Vol 276 (15) ◽  
pp. 11575-11581 ◽  
Author(s):  
Ilana Kogan ◽  
Mohabir Ramjeesingh ◽  
Ling-Jun Huan ◽  
Yanchun Wang ◽  
Christine E. Bear
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Cross Talk ◽  
Nucleotide Binding ◽  
Cystic Fibrosis Gene ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Cystic Fibrosis Transmembrane

Mutations in the cystic fibrosis gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) lead to altered chloride (Cl−) flux in affected epithelial tissues. CFTR is a Cl−channel that is regulated by phosphorylation, nucleotide binding, and hydrolysis. However, the molecular basis for the functional regulation of wild type and mutant CFTR remains poorly understood. CFTR possesses two nucleotide binding domains, a phosphorylation-dependent regulatory domain, and two transmembrane domains that comprise the pore through which Cl−permeates. Mutations of residues lining the channel pore (e.g.R347D) are typically thought to cause disease by altering the interaction of Cl−with the pore. However, in the present study we show that the R347D mutation and diphenylamine-2-carboxylate (an open pore inhibitor) also inhibit CFTR ATPase activity, revealing a novel mechanism for cross-talk from the pore to the catalytic domains. In both cases, the reduction in ATPase correlates with a decrease in nucleotide turnover rather than affinity. Finally, we demonstrate that glutathione (GSH) inhibits CFTR ATPase and that this inhibition is altered in the CFTR-R347D variant. These findings suggest that cross-talk between the pore and nucleotide binding domains of CFTR may be important in thein vivoregulation of CFTR in health and disease.

scholarly journals ATPase Activity of the Cystic Fibrosis Transmembrane Conductance Regulator

1996 ◽  
Vol 271 (45) ◽  
pp. 28463-28468 ◽  
Author(s):  
Canhui Li ◽  
Mohabir Ramjeesingh ◽  
Wei Wang ◽  
Elizabeth Garami ◽  
Marek Hewryk ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

scholarly journals Cystic fibrosis transmembrane conductance regulator: the NBF1+R (nucleotide-binding fold 1 and regulatory domain) segment acting alone catalyses a Co2+/Mn2+/Mg2+-ATPase activity markedly inhibited by both Cd2+ and the transition-state analogue orthovanadate

2003 ◽  
Vol 371 (2) ◽  
pp. 451-462 ◽  
Author(s):  
Jean Philippe ANNEREAU ◽  
Young Hee KO ◽  
Peter L. PEDERSEN
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Transition State ◽  
Nucleotide Binding ◽  
Fusion Partner ◽  
Transmembrane Conductance Regulator ◽  
Regulatory Domain ◽  
Transmembrane Conductance ◽  
Transition State Analogue ◽  
Cystic Fibrosis Transmembrane

Cystic fibrosis (CF) is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator), a regulated anion channel and member of the ATP-binding-cassette transporter (ABC transporter) superfamily. Of CFTR's five domains, the first nucleotide-binding fold (NBF1) has been of greatest interest both because it is the major ‘hotspot’ for mutations that cause CF, and because it is connected to a unique regulatory domain (R). However, attempts have failed to obtain a catalytically active NBF1+R protein in the absence of a fusion partner. Here, we report that such a protein can be obtained following its overexpression in bacteria. The pure NBF1+R protein exhibits significant ATPase activity [catalytic-centre activity (turnover number) 6.7min−1] and an apparent affinity for ATP (Km, 8.7μM) higher than reported previously for CFTR or segments thereof. As predicted, the ATPase activity is inhibited by mutations in the Walker A motif. It is also inhibited by vanadate, a transition-state analogue. Surprisingly, however, the best divalent metal activator is Co2+, followed by Mn2+ and Mg2+. In contrast, Ca2+ is ineffective and Cd2+ is a potent inhibitor. These novel studies, while demonstrating clearly that CFTR's NBF1+R segment can act independently as an active, vanadate-sensitive ATPase, also identify its unique cation activators and a new inhibitor, thus providing insight into the nature of its active site.

scholarly journals Differential regulation of cystic fibrosis transmembrane conductance regulator and Na+,K+-ATPase in gills of striped bass, Morone saxatilis: effect of salinity and hormones

2007 ◽  
Vol 192 (1) ◽  
pp. 249-260 ◽  
Author(s):  
Steffen Søndergaard Madsen ◽  
Lars Nørholm Jensen ◽  
Christian Kølbæk Tipsmark ◽  
Pia Kiilerich ◽  
Russell John Borski
Keyword(s):  
Cystic Fibrosis ◽  
Atpase Activity ◽  
Striped Bass ◽  
Mrna Level ◽  
Mrna Levels ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Α Subunit ◽  
Igf I ◽  
Cystic Fibrosis Transmembrane

Effects of salinity and hormones on cystic fibrosis transmembrane conductance regulator (CFTR) and α-subunit Na+,K+-ATPase (α-NKA) mRNA (analysed by semi-quantitative PCR) and protein expression (analysed by western blotting and immunocytochemistry) were investigated in gills of striped bass. Freshwater (FW) to seawater (SW) transfer induced a disturbance in serum [Na+]. Gill CFTR protein, mRNA level and Na+,K+-ATPase activity were unaffected by SW transfer, whereas α-NKA mRNA increased after transfer. CFTR immunoreactivity was observed in large cells in FW and SW gill filaments at equal intensity. Cortisol decreased serum [Na+] in FW fish, but had no effect on gill Na+,K+-ATPase activity, α-NKA and CFTR mRNA levels. Incubation of gill tissue with cortisol (24 h, >0.01 μg/ml) and epidermal growth factor (EGF 10 μg/ml) decreased CFTR mRNA levels relative to pre-incubation and control levels. CFTR expression was unaffected by IGF-I (10 μg/ml). α-NKA mRNA levels decreased by 50% after 24 h control incubation; it was slightly stimulated by cortisol and unaffected by IGF-I and EGF. In isolated gill cells, phosphorylation of extracellular-regulated kinase (ERK) 1/2 was stimulated by EGF but not affected by IGF-I. This study is the first to report a branchial EGF response and to demonstrate a functional ERK 1/2 pathway in the teleost gill. In conclusion, CFTR and Na+,K+-ATPase are differentially regulated by salinity and hormones in gills of striped bass, despite the putative involvement of both in salt excretion.

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