The cystic fibrosis transmembrane conductance regulator attenuates the endogenous Ca 2+ activated Cl - conductance of Xenopus oocytes

1997 ◽  
Vol 435 (1) ◽  
pp. 178-181 ◽  
Author(s):  
K. Kunzelmann ◽  
M. Mall ◽  
M. Briel ◽  
A. Hipper ◽  
R. Nitschke ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Xenopus Oocytes ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Cl Conductance

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.

Radiotracer studies of cystic fibrosis transmembrane conductance regulator expressed in Xenopus oocytes

1994 ◽  
Vol 266 (6) ◽  
pp. C1586-C1593 ◽  
Author(s):  
T. Ohrui ◽  
W. Skach ◽  
M. Thompson ◽  
J. Matsumoto-Pon ◽  
C. Calayag ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Carboxylic Acid ◽  
Phorbol Ester ◽  
Xenopus Oocytes ◽  
Disulfonic Acid ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Urea Uptake ◽  
Cystic Fibrosis Transmembrane

We measured fluxes of radiotracers in Xenopus oocytes expressing the cystic fibrosis transmembrane conductance regulator (CFTR). Addition of adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents [forskolin and 3-isobutyl-1-methylxanthine (I/F)] led to large increases in uptake of 36Cl, 125I, and 82Br into oocytes expressing wild-type CFTR or delta F508 CFTR but not sham-injected oocytes. I/F also stimulated halide efflux from CFTR and delta F508 oocytes in the sequence Cl > Br > I. cAMP-induced increases in 36Cl efflux from delta F508 oocytes were approximately 20% of those in CFTR oocytes. Increases in halide efflux were blocked by diphenylamine-2-carboxylic acid but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The phorbol ester, phorbol 12-myristate 13-acetate, also stimulated 36Cl efflux from CFTR oocytes. ATP uptakes into CFTR and sham oocytes were similar, and both were reduced by I/F. However, ATP uptake into I/F-treated CFTR oocytes was slightly greater (approximately 40%) than into I/F-treated sham oocytes. Urea uptake into CFTR and sham oocytes was similar and in both cases was increased by I/F. However, the I/F-induced increase in urea uptake into CFTR oocytes was significantly greater than for sham oocytes. I/F stimulated formate uptake into CFTR oocytes but not into sham oocytes. Fluxes of 22Na, 86Rb, 35SO4, 32PO4, and mannitol were unaltered by expression and activation of CFTR.

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.

scholarly journals Cystic Fibrosis Transmembrane Conductance Regulator–associated ATP Release Is Controlled by a Chloride Sensor

1998 ◽  
Vol 143 (3) ◽  
pp. 645-657 ◽  
Author(s):  
Qinshi Jiang ◽  
Daniel Mak ◽  
Sreenivas Devidas ◽  
Erik M. Schwiebert ◽  
Alvina Bragin ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Molecular Mechanisms ◽  
Atp Release ◽  
Chloride Concentration ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Channel Pore ◽  
Cystic Fibrosis Transmembrane ◽  
Atp Efflux ◽  
Cl Conductance

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl− that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl− conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl− conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl >> Br; R347P, Cl >> Br; R347E, Br >> Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl− binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl−. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.

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