Cystic Fibrosis, the CFTR, and Rectifying Cl- Channels

1991 ◽  
pp. 253-272 ◽  
Author(s):  
J. J. Wine ◽  
D. J. Brayden ◽  
G. Hagiwara ◽  
M. E. Krouse ◽  
T. C. Law ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cl Channels

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.

G protein G alpha i-2 inhibits outwardly rectifying chloride channels in human airway epithelial cells

1995 ◽  
Vol 269 (2) ◽  
pp. C451-C456 ◽  
Author(s):  
E. M. Schwiebert ◽  
D. C. Gruenert ◽  
W. B. Guggino ◽  
B. A. Stanton
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
G Protein ◽  
G Proteins ◽  
Airway Epithelial Cells ◽  
Protein G ◽  
Airway Epithelial ◽  
Human Airway ◽  
Cl Channels ◽  
Human Airway Epithelial Cells

Previously we demonstrated that the heterotrimeric G protein, G alpha i-2, inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels in human airway epithelial cells (E. M. Schwiebert, F. Gesek, L. Ercolani, C. Wjasow, D. C. Gruenert, and B. A. Stanton. Am. J. Physiol. 267 (Cell Physiol. 36): C272-C281, 1994, and E. M. Schwiebert, N. L. Kizer, D. C. Gruenert, and B. A. Stanton. Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992). The goal of the present study was to determine if G proteins also regulate outwardly rectifying Cl- channels (ORCC), a distinct class of Cl- channels regulated defectively by protein kinase A (PKA) in cystic fibrosis (CF). To this end, we used the patch-clamp technique to study ORCC in a normal human airway epithelial cell line (9HTEo-) that expresses CFTR and ORCC. Stimulation of G proteins with GTP and GTP gamma S decreased the single-channel open probability (Po) of ORCC, whereas inhibition of G proteins by GDP beta S increased the Po. Moreover, pertussis toxin (PTX), an uncoupler of Gi and G(o) subclasses of heterotrimeric G proteins, also increased the Po. Purified G alpha i-2 decreased the Po. In contrast, other PTX-sensitive G proteins, G alpha i-1, G alpha i-3, and G alpha o, had no effect on Po. We propose that G alpha i-2 couples to a receptor whose agonist negatively regulates ORCC in human airway epithelial cells.

Both the wild type and a functional isoform of CFTR are expressed in kidney

1996 ◽  
Vol 270 (6) ◽  
pp. F1038-F1048 ◽  
Author(s):  
M. M. Morales ◽  
T. P. Carroll ◽  
T. Morita ◽  
E. M. Schwiebert ◽  
O. Devuyst ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Transmembrane Domain ◽  
Renal Medulla ◽  
Regulatory Domain ◽  
Wild Type ◽  
Transmembrane Segments ◽  
Binding Domains ◽  
Cl Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) consists of five domains, two transmembrane-spanning domains, each composed of six transmembrane segments, a regulatory domain, and two nucleotide-binding domains (NBDs). CFTR is expressed in kidney, but its role in overall renal function is not well understood, because mutations in CFTR found in patients with cystic fibrosis are not associated with renal dysfunction. To learn more about the distribution and functional forms of CFTR in kidney, we used a combination of molecular, cell biological, and electrophysiological approaches. These include an evaluation of CFTR mRNA and protein expression, as well as both two-electrode and patch clamping of CFTR expressed either in Xenopus oocytes or mammalian cells. In addition to wild-type CFTR mRNA, an alternate form containing only the first transmembrane domain (TMD), the first NBD, and the regulatory domain (TNR-CFTR) is expressed in kidney. Although missing the second set of TMDs and the second NBD, when expressed in Xenopus oocytes, TNR-CFTR has cAMP-dependent protein kinase A (PKA)-stimulated single Cl- channel characteristics and regulation of PKA activation of outwardly rectifying Cl- channels that are very similar to those of wild-type CFTR. TNR-CFTR mRNA is produced by an unusual mRNA processing mechanism and is expressed in a tissue-specific manner primarily in renal medulla.

CFTR mediates electrogenic chloride secretion in mouse inner medullary collecting duct (mIMCD-K2) cells

1995 ◽  
Vol 269 (3) ◽  
pp. C683-C689 ◽  
Author(s):  
D. Vandorpe ◽  
N. Kizer ◽  
F. Ciampollilo ◽  
B. Moyer ◽  
K. Karlson ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Patch Clamp ◽  
Collecting Duct ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Inner Medullary Collecting Duct ◽  
Cl Channel ◽  
Cl Channels ◽  
Medullary Collecting Duct ◽  
Cystic Fibrosis Transmembrane

Previously we demonstrated that the inner medullary collecting duct cell line mIMCD-K2 secretes Cl- by an electrogenic mechanism [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995; N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F854-F861, 1995]. The goal of the present study was to characterize the Cl- channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl- secretion. To this end, using the patch-clamp technique, we measured Cl- currents. In whole cell patch-clamp experiments, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) activated Cl- currents that were time and voltage independent, inhibited by diphenylamine 2-carboxylate (DPC), and had a linear current-voltage (I-V) relation. In cell-attached patches of the apical membrane, we identified 7-pS Cl- channels that were stimulated by CPT-cAMP. In inside-out patches with Cl- in the pipette and bath solutions, Cl- currents had a linear I-V relation. The halide permeability sequence was PCl = PBr > PI. The Cl- channel inhibitors DPC, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and glibenclamide blocked the 7-pS Cl- channel, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was ineffective. By reverse transcriptase polymerase chain reaction, we isolated a partial cDNA clone encoding the cystic fibrosis transmembrane conductance regulator in mIMCD-K2 cells. We conclude that cAMP stimulates electrogenic Cl- secretion in inner medullary collecting duct cells by activating cystic fibrosis transmembrane conductance regulator Cl- channels.

Long-term cAMP activation of Na(+)-K(+)-2Cl- cotransporter activity in HT-29 human adenocarcinoma cells

1993 ◽  
Vol 264 (4) ◽  
pp. C857-C865 ◽  
Author(s):  
I. N. Slotki ◽  
W. V. Breuer ◽  
R. Greger ◽  
Z. I. Cabantchik
Keyword(s):  
Cystic Fibrosis ◽  
Cell Lines ◽  
De Novo ◽  
Transport Systems ◽  
Short Term ◽  
Cl Channel ◽  
Epithelial Cell Lines ◽  
Cl Channels ◽  
Ht 29

Cl- channel and Na(+)-K(+)-2Cl- cotransport activities were studied in various cystic fibrosis transmembrane conductance regulator (CFTR)-expressing cells with the aim of assessing integrative patterns of regulation of Cl- secretion. Human colonic HT-29 cells express relatively high levels of CFTR and cotransporter but relatively low Cl- channel activity. These cells showed commensurate activations of both transport systems evoked by short-term (minutes) or long-term (hours) exposures to adenosine 3',5'-cyclic monophosphate (cAMP). However, unlike in the case of CFTR and Cl- channels, long-term induction of cotransporter did not depend on de novo protein synthesis or changes in number of transporters. The patterns of activation of both transporters were also examined in CFTR-deficient cell lines (CFPAC and the viral-transfected CFPAC-PLJ) and in the viral CFTR-transfected derivative (CFPAC-4.7). All these cells displayed relatively low basal cotransport activity and a correspondingly low number of transporters, whereas only CFPAC-4.7 cells showed short-term (but not long-term) activatable Cl- channels. However, irrespective of the presence or absence of CFTR in CFPAC cells, neither short- nor long-term cAMP exposures induced significant cotransporter activation. Our studies with the various epithelial cell lines indicate that expression of CFTR activity per se is not sufficient for stimulation of cotransporter activity. Moreover, despite apparent correction of CFTR levels in CFPAC cells by gene transfer, the apparent Cl- secretory capacity might be limited by the low cotransport activity, such as that found in CFPAC cells, with obvious implications for proposed gene therapy of cystic fibrosis.

scholarly journals Cystic fibrosis gene expression is not correlated with rectifying Cl- channels.

1991 ◽  
Vol 88 (12) ◽  
pp. 5277-5281 ◽  
Author(s):  
C. L. Ward ◽  
M. E. Krouse ◽  
D. C. Gruenert ◽  
R. R. Kopito ◽  
J. J. Wine
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Cystic Fibrosis Gene ◽  
Cl Channels

scholarly journals Regulation of Cl- channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP.

1992 ◽  
Vol 89 (5) ◽  
pp. 1621-1625 ◽  
Author(s):  
M. J. Stutts ◽  
T. C. Chinet ◽  
S. J. Mason ◽  
J. M. Fullton ◽  
L. L. Clarke ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Extracellular Atp ◽  
Airway Epithelial ◽  
Cystic Fibrosis Airway ◽  
Cl Channels

Modulation of Cl- secretion by benzimidazolones. II. Coordinate regulation of apical GCl and basolateral GK

1996 ◽  
Vol 271 (5) ◽  
pp. L785-L795 ◽  
Author(s):  
D. C. Devor ◽  
A. K. Singh ◽  
R. J. Bridges ◽  
R. A. Frizzell
Keyword(s):  
Cystic Fibrosis ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Secretory Response ◽  
K Channel ◽  
T84 Cells ◽  
Coordinate Regulation ◽  
Cl Secretion ◽  
Cl Channels ◽  
Subsequent Addition

We previously demonstrated that the novel benzimidazolone, 1-ethyl-2-benzimidazolinone (1-EBIO), stimulates a sustained Cl- secretory response across T84 monolayers by opening a Ca(2+)-dependent basolateral K+ channel. In the present work, we evaluated the effects on Cl-secretion of other benzimidazolones, NS-004 and NS-1619, which have been shown to open cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast to 1-EBIO, neither NS-004 nor NS-1619 stimulated a significant Cl- secretory current (Isc). Neither NS-004 nor NS-1619 increased Isc subsequent to forskolin stimulation. However, when added after 1-EBIO, NS-004 and NS-1619 stimulated large sustained increases in Isc. In addition, NS-004 and NS-1619 potentiated the effects of carbachol. We used nystatin to permeabilize the apical or basolateral membrane to determine the effects of NS-004 and 1-EBIO on the basolateral K+ (IK) and apical Cl- (ICl) currents. Both NS-004 and 1-EBIO increased ICl, and the stimulated currents were inhibited by glibenclamide. In contrast, NS-004 failed to significantly affect IK, but subsequent addition of 1-EBIO induced a large increase in IK. The effects of 1-EBIO, NS-004, and NS-1619 on the Ca(2+)-dependent K+ channel (KCa) in T84 cells was determined in excised inside-out patches. Neither NS-004 nor NS-1619 affected K+ channel activity, whereas the subsequent addition of 1-EBIO produced a marked channel activation. Results similar to those observed in T84 monolayers were obtained from murine airway cell primary cultures: NS-004 or NS-1619 had no effect on Isc, whereas 1-EBIO stimulated a sustained Cl- secretory response. The results demonstrate that activation of CFTR alone is insufficient to evoke transepithelial Cl- secretion. Activation of the basolateral membrane K+ channel is a necessary component of the secretory response. Thus the basolateral membrane KCa may be a novel pharmacological target in cystic fibrosis therapy.

Swelling-induced and depolarization-induced C1-channels in normal and cystic fibrosis epithelial cells

1991 ◽  
Vol 261 (4) ◽  
pp. C658-C674 ◽  
Author(s):  
C. K. Solc ◽  
J. J. Wine
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Current Density ◽  
Single Channel ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Channel Activity ◽  
Whole Cell ◽  
Cl Channels ◽  
Single Channel Currents

Cl- currents induced by cell swelling were characterized at the whole cell and single-channel levels in primary cultures of normal and cystic fibrosis (CF) epithelial cells and in the T84 cell line. Currents recorded in normal and CF cells were indistinguishable. At 22-24 degrees C with isotonic CsCl in the pipette, initial whole cell outward current density at 100 mV in unswollen cells was 2-4 pA/pF. The current density increased with time during whole cell recording up to 100 pA/pF in isotonic solutions and up to 200 pA/pF in a hypotonic bath, though values typically ranged between 10 and 70 pA/pF. Currents were outwardly rectifying, active at negative voltages, started to inactivate above approximately 40 mV, and were blocked by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Single Cl- channels (approximately 50 pS near 0 mV) with an outwardly rectifying current-voltage relation were recorded in cell-attached and outside-out patches from swollen cells. The channels were mostly open at negative voltages and inactivated at positive voltages with a voltage dependence similar to the whole cell currents. Channel activity decreased rapidly (channel rundown) after seal formation. After swelling-induced channel activity had ceased, outwardly rectifying, depolarization-induced Cl- channels (ORDIC channels) were activated in some patches. The swelling-induced and ORDIC single-channel currents were similar, but some consistent differences were observed. ORDIC channels were often closed at resting voltages (-70 to -50 mV), while swelling-induced channels were always open in this voltage range. In addition, ORDIC channels started to inactivate at more positive voltages (approximately 90 vs. approximately 50 mV), rectified more, and had smaller conductances (approximately 25 pS near 0 mV), shorter mean open durations (approximately 70 vs. approximately 350 ms), and more open-channel noise than swelling-induced channels. The two types of currents might arise from separate channel proteins or from a single channel molecule in different states.

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