Hydrolytic and nonhydrolytic interactions in the ATP regulation of CFTR Cl- conductance

1996 ◽  
Vol 271 (1) ◽  
pp. C35-C42 ◽  
Author(s):  
M. M. Reddy ◽  
P. M. Quinton
Keyword(s):  
Cystic Fibrosis ◽  
Significant Role ◽  
Atp Hydrolysis ◽  
Transmembrane Conductance Regulator ◽  
Sweat Duct ◽  
Transmembrane Conductance ◽  
Atp Regulation ◽  
Cystic Fibrosis Transmembrane ◽  
Cl Conductance

Previously, we showed in the native sweat duct that, in the presence of 0.1-0.5 mM ATP, nonhydrolyzable ATP analogue adenosine 5'-adenylylimidodiphosphate (AMP-PNP) can activate cystic fibrosis transmembrane conductance regulator Cl- conductance (CFTR GCl) (15). The objective of this study is to determine if 1) nonhydrolytic ATP binding alone can activate CFTR GCl after stable phosphorylation [in the presence of adenosine 5'-O-(3-thiotriphosphate) and phosphatase inhibition cocktail] of CFTR or 2) an ATP hydrolysis (in addition to phosphorylation) is required to support subsequent nonhydrolytic ATP regulation of CFTR GCl. We show that stably phosphorylated CFTR could only be activated by AMP-PNP in the presence of a small background ATP concentration. However, AMP-PNP can sustain previously activated CFTR GCl in the absence of ATP, even though Mg2+ is required for phosphorylation activation of CFTR GCl. However, once stably phosphorylated, ATP activation of CFTR GCl is independent of Mg2+. Our results show that both hydrolytic and nonhydrolytic interactions regulate CFTR GCl in vivo. Nonhydrolytic ATP interaction plays a significant role in both activation and deactivation of CFTR GCl.

Deactivation of CFTR-Cl conductance by endogenous phosphatases in the native sweat duct

1996 ◽  
Vol 270 (2) ◽  
pp. C474-C480 ◽  
Author(s):  
M. M. Reddy ◽  
P. M. Quinton
Keyword(s):  
Cystic Fibrosis ◽  
Okadaic Acid ◽  
Apical Membrane ◽  
Transmembrane Conductance Regulator ◽  
Sweat Duct ◽  
Transmembrane Conductance ◽  
Phosphatase Inhibitors ◽  
Cystic Fibrosis Transmembrane ◽  
Cl Conductance

Cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated Cl channel. However, very little is known about the endogenous mechanism(s) of deactivation of CFTR-Cl conductance (CFTR-GCl) in vivo. We studied the action of endogenous phosphatases in regulation of the adenosine 3',5'-cyclic monophosphate (cAMP)- and ATP-induced CFTR-GCl in the apical membrane of microperfused preparations of basolaterally permeabilized native sweat duct. Activation of CFTR-GCl was monitored by measuring the apical Cl diffusion potentials and GCl, which spontaneously deactivated on removal of cAMP. This spontaneous loss of CFTR-GCl activity could be prevented by a cocktail of phosphatase inhibitors (fluoride, vanadate, and okadaic acid). We studied the effects of each of these phosphatase antagonists on the rate of deactivation of CFTR-GCl after cAMP washout. In contrast to vanadate or fluoride, okadaic acid virtually prevented deactivation of CFTR-GCl after cAMP washout. We conclude that either or both protein phosphatases 1 and 2A are responsible for the dephosphorylation deactivation of CFTR-GCl in vivo.

Examining basal chloride transport using the nasal potential difference response in a murine model

2001 ◽  
Vol 281 (5) ◽  
pp. L1173-L1179 ◽  
Author(s):  
Kristine G. Brady ◽  
Thomas J. Kelley ◽  
Mitchell L. Drumm
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Transport ◽  
Inbred Mice ◽  
Inbred Strains ◽  
Potential Difference ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Inbred Strains Of Mice ◽  
Cystic Fibrosis Transmembrane

Epithelia of humans and mice with cystic fibrosis are unable to secrete chloride in response to a chloride gradient or to cAMP-elevating agents. Bioelectrical properties measured using the nasal transepithelial potential difference (TEPD) assay are believed to reflect these cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride transport defects. Although the response to forskolin is CFTR mediated, the mechanisms responsible for the response to a chloride gradient are unknown. TEPD measurements performed on inbred mice were used to compare the responses to low chloride and forskolin in vivo. Both responses show little correlation between or within inbred strains of mice, suggesting they are mediated through partially distinct mechanisms. In addition, these responses were assayed in the presence of several chloride channel inhibitors, including DIDS, diphenylamine-2-carboxylate, glibenclamide, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and a protein kinase A inhibitor, the Rp diastereomer of adenosine 3′,5′-cyclic monophosphothioate ( Rp-cAMPS). The responses to low chloride and forskolin demonstrate significantly different pharmacological profiles to both DIDS and Rp-cAMPS, indicating that channels in addition to CFTR contribute to the low chloride response.

Biosynthesis and Degradation of CFTR

1999 ◽  
Vol 79 (1) ◽  
pp. S167-S173 ◽  
Author(s):  
RON R. KOPITO
Keyword(s):  
Cystic Fibrosis ◽  
Secretory Pathway ◽  
Covalent Modification ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
The Common ◽  
Effects Of Temperature ◽  
Cystic Fibrosis Transmembrane

Kopito, Ron R. Biosynthesis and Degradation of CFTR. Physiol. Rev. 79, Suppl.: S167–S173, 1999. — Many of the mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that cause cystic fibrosis interfere with the folding and biosynthetic processing of nascent CFTR molecules in the endoplasmic reticulum. Mutations in the cytoplasmic nucleotide binding domains, including the common allele ΔF508, decrease the efficiency of CFTR folding, reduce the probability of its dissociation from molecular chaperones, and largely prevent its maturation through the secretory pathway to the plasma membrane. These mutant CFTR molecules are rapidly degraded by cytoplasmic proteasomes by a process that requires covalent modification by multiubiquitination. The effects of temperature and chemical chaperones on the intracellular processing of mutant CFTR molecules suggest that strategies aimed at increasing the folding yield of this protein in vivo may eventually lead to the development of novel therapies for cystic fibrosis.

scholarly journals Inhibition of ATPase, GTPase and adenylate kinase activities of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator by genistein

1999 ◽  
Vol 340 (1) ◽  
pp. 227-235 ◽  
Author(s):  
Christoph RANDAK ◽  
Ennes A. AUERSWALD ◽  
Irmgard ASSFALG-MACHLEIDT ◽  
William W. REENSTRA ◽  
Werner MACHLEIDT
Keyword(s):  
Cystic Fibrosis ◽  
Adenylate Kinase ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Enhanced Fluorescence ◽  
Chloride Currents ◽  
Cystic Fibrosis Transmembrane ◽  
Ordered State

In the presence of ATP, genistein, like the ATP analogue adenosine 5ʹ-[β,γ-imido]triphosphate (pp[NH]pA), increases cystic fibrosis transmembrane conductance regulator (CFTR) chloride currents by prolonging open times. As pp[NH]pA is thought to increase CFTR currents by interfering with ATP hydrolysis at the second nucleotide-binding fold (NBF-2), the present study was undertaken to investigate the effects of genistein on a fusion protein comprising maltose-binding protein (MBP) and NBF-2 (MBP-NBF-2). MBP-NBF-2 exhibited ATPase, GTPase and adenylate kinase activities that were inhibited by genistein in a partial non-competitive manner with respect to ATP or GTP. Ki values for competitive and uncompetitive inhibition were respectively 20 μM and 63 μM for ATPase, 15 μM and 54 μM for GTPase, and 46 μM and 142 μM for adenylate kinase. For ATPase activity, genistein reduced Vmax by 29% and Vmax/Km by 77%. Additional evidence for complex-formation between genistein and MBP-NBF-2 was obtained by the detection of genistein-dependent alterations in the CD spectrum of MBP-NBF-2 that were consistent with the formation of a higher-ordered state. Addition of MBP-NBF-2 increased the fluorescence intensity of genistein, consistent with a change to a less polar environment. pp[NH]pA partially eliminated this enhanced fluorescence of genistein. These observations provide the first direct biochemical evidence that genistein interacts with CFTR, thus inhibiting NBF-2 activity, and suggest a similar mechanism for genistein-dependent stimulation of CFTR chloride currents.

scholarly journals In vivo crystals reveal critical features of the interaction between cystic fibrosis transmembrane conductance regulator (CFTR) and the PDZ2 domain of Na+/H+ exchange cofactor NHERF1

2020 ◽  
Vol 295 (14) ◽  
pp. 4464-4476
Author(s):  
Eleanor R. Martin ◽  
Alessandro Barbieri ◽  
Robert C. Ford ◽  
Robert C. Robinson
Keyword(s):  
Cystic Fibrosis ◽  
Protein Interactions ◽  
Pdz Domain ◽  
Binding Motif ◽  
Transmembrane Conductance Regulator ◽  
Protein Protein Interactions ◽  
Reporter System ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

Crystallization of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under extremely nonphysiological protein, salt, and H+ concentrations. Here, we describe the development of a robust Inka1-Box (iBox)–PAK4cat system that spontaneously crystallizes in several mammalian cell types. The semi-quantitative assay described here allows the measurement of in vivo protein-protein interactions using a novel GFP-linked reporter system that produces fluorescent readouts from protein crystals. We combined this assay with in vitro X-ray crystallography and molecular dynamics studies to characterize the molecular determinants of the interaction between the PDZ2 domain of Na+/H+ exchange regulatory cofactor NHE-RF1 (NHERF1) and cystic fibrosis transmembrane conductance regulator (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis. These experiments revealed the crystal structure of the extended PDZ domain of NHERF1 and indicated, contrary to what has been previously reported, that residue selection at positions −1 and −3 of the PDZ-binding motif influences the affinity and specificity of the NHERF1 PDZ2-CFTR interaction. Our results suggest that this system could be utilized to screen additional protein-protein interactions, provided they can be accommodated within the spacious iBox-PAK4cat lattice.

scholarly journals Adenosine Triphosphate–dependent Asymmetry of Anion Permeation in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

1998 ◽  
Vol 111 (4) ◽  
pp. 601-614 ◽  
Author(s):  
Paul Linsdell ◽  
John W. Hanrahan
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channel ◽  
Atp Hydrolysis ◽  
Organic Anions ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Extracellular Solution ◽  
Anion Permeation ◽  
Atpase Inhibitors ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a tightly regulated channel that mediates the passive diffusion of Cl− ions. Here we show, using macroscopic current recording from excised membrane patches, that CFTR also shows significant, but highly asymmetrical, permeability to a broad range of large organic anions. Thus, all large organic anions tested were permeant when present in the intracellular solution under biionic conditions (PX/PCl = 0.048–0.25), whereas most were not measurably permeant when present in the extracellular solution. This asymmetry was not observed for smaller anions. ATPase inhibitors that “lock” CFTR channels in the open state (pyrophosphate, 5′-adenylylimidodiphosphate) disrupted the asymmetry of large anion permeation by allowing their influx from the extracellular solution, which suggests that ATP hydrolysis is required to maintain asymmetric permeability. The ability of CFTR to allow efflux of large organic anions represents a novel function of CFTR. Loss of this function may contribute to the pleiotropic symptoms seen in cystic fibrosis.

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