Genistein activates CFTR Cl- channels via a tyrosine kinase- and protein phosphatase-independent mechanism

1997 ◽  
Vol 273 (2) ◽  
pp. C747-C753 ◽  
Author(s):  
P. J. French ◽  
J. Bijman ◽  
A. G. Bot ◽  
W. E. Boomaars ◽  
B. J. Scholte ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Direct Interaction ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cl Channels ◽  
Dependent Protein ◽  
Cystic Fibrosis Transmembrane

Previous studies have revealed an adenosine 3',5'-cyclic monophosphate (cAMP)-independent activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by the tyrosine kinase inhibitor genistein. To further explore its mechanism of action, we have reconstituted genistein activation of CFTR in excised inside-out membrane patches. In the presence or absence of ATP, genistein appeared unable to open silent CFTR Cl- channels. However, on CFTR prephosphorylation by cAMP-dependent protein kinase (cAK), genistein enhanced CFTR activity by twofold, resulting from a prolonged burst duration. Genistein could also hyperactivate partially phosphorylated CFTR in the absence of cAK and therefore is different from 5'-adenylylimidodiphosphate, which required fully phosphorylated CFTR. Phosphatase-resistant thiophosphorylation likewise primed the CFTR Cl- channel for hyperactivation by genistein in the absence of cAK. Replacement of ATP by GTP as a hydrolyzable nucleotide triphosphate for CFTR did not impair the ability of genistein to activate thiophosphorylated CFTR, despite the fact that GTP is a poor substrate for tyrosine kinases. These findings argue against a role of protein phosphatases or tyrosine kinases but suggest a more direct interaction of genistein with CFTR, possibly at the level of the second nucleotide-binding domain.

CFTR chloride channel activation by genistein: the role of serine/threonine protein phosphatases

1996 ◽  
Vol 271 (2) ◽  
pp. C650-C657 ◽  
Author(s):  
W. W. Reenstra ◽  
K. Yurko-Mauro ◽  
A. Dam ◽  
S. Raman ◽  
S. Shorten
Keyword(s):  
Chloride Channel ◽  
Kinase Inhibitor ◽  
Maximal Activity ◽  
Calyculin A ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Nih 3T3 ◽  
Cystic Fibrosis Transmembrane

We have previously shown [B. Illek, H. Fischer, G. F. Santos, J. H. Widdicombe, T. E. Machen, and W. W. Reenstra, Am. J. Physiol. 268 (Cell Physiol. 37): C886-C893, 1995] that genistein, a tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in NIH/3T3 cells that have been stably transfected with an expression vector for the CFTR (NIH-CFTR cells). In this study, we present evidence suggesting that both genistein and the serine/threonine protein phosphatase (PPase) inhibitor calyculin A activate the CFTR by inhibiting PPase activity. As measured by 125I efflux, genistein and calyculin A stimulate the CFTR to approximately 50% of the maximal activity with forskolin. Neither agonist increases CFTR activity at saturating forskolin concentrations, but genistein and calyculin A have an additive effect on CFTR activity. Forskolin, but neither genistein nor calyculin A, stimulates protein kinase A(PKA) activity. The PKA inhibitor H-89 inhibits CFTR activation and in vivo phosphorylation by all three agonists. Proteolytic digestion of in vivo phosphorylated CFTR suggests that the CFTR is phosphorylated on the same sites during stimulation with genistein and forskolin but on different sites stimulation with calyculin A. The data suggest that genistein and calyculin A inhibit different PPase activities, allowing CFTR phosphorylation and partial stimulation, by a basal PKA activity.

Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia

2000 ◽  
Vol 279 (5) ◽  
pp. L835-L841 ◽  
Author(s):  
Olafur Baldursson ◽  
Herbert A. Berger ◽  
Michael J. Welsh
Keyword(s):  
Functional Role ◽  
Transmembrane Conductance Regulator ◽  
Regulatory Domain ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Dependent Protein ◽  
Rat Thyroid ◽  
Cystic Fibrosis Transmembrane

The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl− current. Mutation of four in vivo phosphorylation sites, Ser660, Ser737, Ser795, and Ser813 (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser660 or Ser813 alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser660 nor Ser813 alone increased current to wild-type levels; both residues were required. Changing Ser737 to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser737 may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.

PKC-sensitive Cl- channels associated with ciliary epithelial homologue of pICln

1995 ◽  
Vol 268 (3) ◽  
pp. C572-C579 ◽  
Author(s):  
M. Coca-Prados ◽  
J. Anguita ◽  
M. L. Chalfant ◽  
M. M. Civan
Keyword(s):  
Northern Analysis ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Current Voltage ◽  
Cl Channels ◽  
Polymerase Chain ◽  
Reaction Cloning ◽  
Cystic Fibrosis Transmembrane ◽  
Nonpigmented Ciliary Epithelial ◽  
Current Voltage Relationship

Swelling activates and protein kinase C (PKC) downregulates Cl- channels in cultured nonpigmented ciliary epithelial (NPE) cells. We now report that the PKC inhibitor staurosporine upregulates whole cell Cl- currents isosmotically. The kinetics and current-voltage relationship are similar to those of volume-activated Cl- channels of these cells. These properties are inconsistent with cloned ClC-0, ClC-1, ClC-2, and MDR1 channels but could reflect the cystic fibrosis transmembrane conductance regulator (CFTR) channel or the Cl- channel regulator pICln. CFTR mRNA was undetectable by Northern analysis of cultured NPE cells or ciliary body tissue. In contrast, a human pICln probe obtained by polymerase chain reaction cloning and showing 90% identity with the rat cDNA clone detected high levels of transcripts in NPE cells. The level was low in tissue, where the NPE message was diluted by RNA from other cells. We conclude that NPE cells display staurosporine-activated Cl- channels [gSt(Cl)] likely identical with the volume-activated channels. The same cells expressing gSt(Cl) transcribe mRNA for a novel homologue (pHCBICln) of pICln that may regulate Cl- transport into the aqueous humor.

scholarly journals Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.

1992 ◽  
Vol 100 (4) ◽  
pp. 573-591 ◽  
Author(s):  
D N Sheppard ◽  
M J Welsh
Keyword(s):  
Cystic Fibrosis ◽  
Voltage Dependence ◽  
K Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
K Channels ◽  
Intracellular Atp ◽  
Cl Channel ◽  
Cl Channels ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by cAMP-dependent phosphorylation and by intracellular ATP. Intracellular ATP also regulates a class of K+ channels that have a distinct pharmacology: they are inhibited by sulfonylureas and activated by a novel class of drugs called K+ channel openers. In search of modulators of CFTR Cl- channels, we examined the effect of sulfonylureas and K+ channel openers on CFTR Cl- currents in cells expressing recombinant CFTR. The sulfonylureas, tolbutamide and glibenclamide, inhibited whole-cell CFTR Cl- currents at half-maximal concentrations of approximately 150 and 20 microM, respectively. Inhibition by both agents showed little voltage dependence and developed slowly; > 90% inhibition occurred 3 min after adding 1 mM tolbutamide or 100 microM glibenclamide. The effect of tolbutamide was reversible, while that of glibenclamide was not. In contrast to their activating effect on K+ channels, the K+ channel openers, diazoxide, BRL 38227, and minoxidil sulfate inhibited CFTR Cl- currents. Half-maximal inhibition was observed at approximately 250 microM diazoxide, 50 microM BRL 38227, and 40 microM minoxidil sulfate. The rank order of potency for inhibition of CFTR Cl- currents was: glibenclamide < BRL 38227 approximately equal to minoxidil sulfate > tolbutamide > diazoxide. Site-directed mutations of CFTR in the first membrane-spanning domain and second nucleotide-binding domain did not affect glibenclamide inhibition of CFTR Cl- currents. However, when part of the R domain was deleted, glibenclamide inhibition showed significant voltage dependence. These agents, especially glibenclamide, which was the most potent, may be of value in identifying CFTR Cl- channels. They or related analogues might also prove to be of value in treating diseases such as diarrhea, which may involve increased activity of the CFTR Cl- channel.

Actin filament organization is required for proper cAMP-dependent activation of CFTR

1999 ◽  
Vol 277 (6) ◽  
pp. C1160-C1169 ◽  
Author(s):  
Adriana G. Prat ◽  
C. Casey Cunningham ◽  
G. Robert Jackson ◽  
Steven C. Borkan ◽  
Yihan Wang ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Single Channel ◽  
Fold Increase ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Whole Cell ◽  
Excised Patches ◽  
Cystic Fibrosis Transmembrane ◽  
Molecular Nature

Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(−)]. cAMP stimulation of ABP(−) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl− currents. In ABP(−) cells expressing CFTR, cAMP was also without effect on Cl− conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl− currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(−)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl−channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl− channel activity in excised patches of ABP(−)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.

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