Abstract
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 the in vivo regulation of CFTR in health and disease.
Highlights
Cystic fibrosis (CF)1 is a genetic, lethal disease that affects epithelial cells lining the duct-like passages of the respiratory tract, gastrointestinal tract, and the biliary and reproductive systems [1]
A modest decrease in ATPase activity (ϳ6%) in phosphorylated CFTR samples was observed with 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), another channel blocker from the arylaminobenzolate family
DPC did not have a major effect on ATP binding to the nucleotide binding domains (NBDs) of phosphorylated CFTR, with a Km (MgATP) (0.4 mM) close to that typically observed in the absence of drug (0.3 mM; [18])
Summary
Production and Purification of CFTR-His Proteins—Procedures describing productions and purification of CFTR-His proteins were published previously [36]. The concentrated CFTR fractions and lipid control were dialyzed in a Spectra/Por dialysis membrane (Spectrum Laboratories Inc., Rancho Dominguez, CA; molecular mass cut off 50,000 Da) overnight at 4 °C against 4 liters of a buffer containing 8 mM Hepes, 0.5 mM EGTA, pH 7.2. PKA was removed after phosphorylation by dialyzing all samples in a Specta/Por dialysis membrane (molecular mass cut off 50,000 Da) overnight at 4 °C against 4 liters of a buffer containing 8 mM Hepes, 0.5 mM EGTA, and 0.025% NaN3, pH 7.2. The ATPase assay was carried out in a reaction mixture containing 30 l of freshly dialyzed proteoliposomes or liposomes, 1 mM nonradioactive ATP, 20 mM Tris, 40 mM NaCl, 4 mM MgCl2, pH 7.5, and 2 Ci of [␣-32P]ATP (10 Ci/l; Amersham Pharmacia Biotech). For comparison of drug treatment to 100% control, onepopulation Student’s t test was used
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