Properties of cAMP-dependent and Ca(2+)-dependent whole cell Cl- conductances in rat epididymal cells

1993 ◽  
Vol 264 (4) ◽  
pp. C794-C802 ◽  
Author(s):  
S. J. Huang ◽  
W. O. Fu ◽  
Y. W. Chung ◽  
T. S. Zhou ◽  
P. Y. Wong
Keyword(s):  
Cystic Fibrosis ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Whole Cell ◽  
Cyclic Monophosphate ◽  
Cation Current ◽  
Whole Cell Patch Clamp ◽  
Current Voltage ◽  
Clamp Condition ◽  
Cl Conductance

Single rat epididymal cell studied under whole cell patch-clamp condition responded to 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) (500 microM) and to ionomycin (1 microM) by an increase in whole cell conductance. A major part of the stimulated current was carried by Cl-, although a small part was due to nonselective cation current. After elimination of the cation current component by using impermeant cation, the cells revealed different Cl- conductance properties in response to adenosine 3',5'-cyclic monophosphate (cAMP) and ionomycin. The cAMP-stimulated Cl- conductance was independent of time and voltage and showed a linear current-voltage relationship. The anion permselectivity was NO3- > Br- > Cl- approximately I- >> SO(4)2-. The ionomycin-stimulated Cl- conductance showed marked time and voltage dependency. In contrast to the cAMP-induced anion permselectivity, the ionomycin-induced anion permselectivity was I- > Br- approximately NO3- > Cl- >> SO(4)2-. These results indicate that the epididymal epithelial cells exhibit different Cl- conductances sensitive to cAMP and Ca2+. The cAMP-activated conductance has properties resembling the type associated with the cystic fibrosis transmembrane conductance regulator found in cystic fibrosis-affected epithelia. This finding supports the notion that the epididymis is a cystic fibrosis epithelium.

In vivo activation of CFTR-dependent chloride transport in murine airway epithelium by CNP

1997 ◽  
Vol 273 (5) ◽  
pp. L1065-L1072 ◽  
Author(s):  
Thomas J. Kelley ◽  
Calvin U. Cotton ◽  
Mitchell L. Drumm
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Transport ◽  
Nasal Epithelium ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Cyclic Monophosphate ◽  
Δf508 Cftr ◽  
Membrane Bound

Inhibitors of guanosine 3′,5′-cyclic monophosphate (cGMP)-inhibited phosphodiesterases stimulate Cl− transport across the nasal epithelia of cystic fibrosis mice carrying the ΔF508 mutation [cystic fibrosis transmembrane conductance regulator (CFTR) (ΔF/ΔF)], suggesting a role for cGMP in regulation of epithelial ion transport. Here we show that activation of membrane-bound guanylate cyclases by C-type natriuretic peptide (CNP) stimulates hyperpolarization of nasal epithelium in both wild-type and ΔF508 CFTR mice in vivo but not in nasal epithelium of mice lacking CFTR [CFTR(−/−)]. With the use of a nasal transepithelial potential difference (TEPD) assay, CNP was found to hyperpolarize lumen negative TEPD by 6.1 ± 0.6 mV in mice carrying wild-type CFTR. This value is consistent with that obtained with 8-bromoguanosine 3′,5′-cyclic monophosphate (6.2 ± 0.9 mV). A combination of the adenylate cyclase agonist forskolin and CNP demonstrated a synergistic ability to induce Cl− secretion across the nasal epithelium of CFTR(ΔF/ΔF) mice. No effect on TEPD was seen with this combination when used on CFTR(−/−) mice, implying that the CNP-induced change in TEPD in CFTR(ΔF/ΔF) mice is CFTR dependent.

Characterization of a swelling-induced chloride conductance in cultured rat epididymal cells

1993 ◽  
Vol 265 (4) ◽  
pp. C997-C1005 ◽  
Author(s):  
H. C. Chan ◽  
W. O. Fu ◽  
Y. W. Chung ◽  
S. J. Huang ◽  
T. S. Zhou ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Induced Current ◽  
Whole Cell ◽  
Cyclic Monophosphate ◽  
Whole Cell Patch Clamp ◽  
Current Activation ◽  
Ethanesulfonic Acid ◽  
Cl Conductance

Swelling-induced Cl- conductance in cultured rat epididymal cells was characterized using whole cell patch-clamp techniques. Activation of whole cell current with an outwardly rectifying current-potential relationship was observed in cells exposed to hyposmotic solutions. This current was determined, from the observed current-reversal potentials at different Cl- concentrations, to be Cl- selective. The anion selectivity sequence of the swelling-induced Cl- conductance was I- approximately NO3- approximately Br- > Cl- > 2-(N-morpholino)ethanesulfonic acid. The swelling-induced Cl- conductance was reversibly inhibited by different Cl- channel blockers. Unlike diphenylamine-2-carboxylate or 5-nitro-2-(3-phenylpropylamino)-benzoate, which showed voltage-independent blockade, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid showed a marked voltage-dependent blockade of the volume-sensitive Cl- current, with a greater effect at depolarizing voltages. The swelling-induced Cl- conductance appeared to be different from the Ca(2+)- or adenosine 3',5'-cyclic monophosphate-activated Cl- conductances on the basis of the following observations: 1) swelling-induced current activation was seen even in the presence of kinase inhibitor (H-8) or absence of external free Ca2+, and 2) further increase in current activation could be produced by swelling after Ca(2+)- or adenosine 3',5'-cyclic monophosphate-induced current activation. The swelling-induced Cl- conductance may be involved in regulating epithelial cell volume as well as serving other important epididymal functions such as facilitating transepithelial secretion of organic compounds.

Rectification of whole cell cystic fibrosis transmembrane conductance regulator chloride current

1995 ◽  
Vol 268 (3) ◽  
pp. C636-C646 ◽  
Author(s):  
J. L. Overholt ◽  
A. Saulino ◽  
M. L. Drumm ◽  
R. D. Harvey
Keyword(s):  
Cystic Fibrosis ◽  
Theory Model ◽  
Rate Theory ◽  
Dependent Manner ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Concentration Dependent Manner ◽  
Whole Cell ◽  
Multiple Binding Sites ◽  
Cystic Fibrosis Transmembrane

Whole cell epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents exhibited a linear current-voltage (I-V) relationship with high symmetrical transmembrane Cl- concentrations. However, when intracellular Cl- (Cli-) was reduced by replacement with glutamate, I-V relationships were outwardly rectifying. Rectification was not affected by reducing extracellular Cl- to eliminate or reverse the gradient, indicating that rectification is not a function of the Cl- gradient. Rectification was affected by Cli- in a concentration-dependent manner, and it was weaker when Cli- was reduced by replacement with sucrose. These characteristics are identical to those of the cardiac isoform of CFTR, and the experimental data could be simulated by an Eyring rate theory model assuming that permeating anions interact at a single binding site within the channel pore. No evidence was found for multiple binding sites. These results indicate that rectification is a function of the concentration and permeability of the anions inside the cell. It is concluded that rectification of CFTR Cl- current is a property of ion channel permeation that would occur under physiological conditions and that permeation of the epithelial and cardiac isoforms of CFTR is identical.

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.

Activation of Cl− currents by intracellular chloride in fibroblasts stably expressing the human cystic fibrosis transmembrane conductance regulator

1993 ◽  
Vol 71 (9) ◽  
pp. 645-649 ◽  
Author(s):  
Xiaodong Wang ◽  
Yoshinori Marunaka ◽  
Ludwik Fedorko ◽  
Sascha Dho ◽  
J. Kevin Foskett ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cyclic Amp ◽  
Patch Clamp ◽  
Single Channel ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Whole Cell ◽  
Cystic Fibrosis Transmembrane

The Cl− conductance of a mouse fibroblast cell line (LTK− cells) that was stably transfected with the human CFTR (cystic fibrosis transmembrane conductance regulator) complementary DNA was studied. Single Cl− channel activity was observed only after treatment of the cells with forskolin, the single-channel conductance being 6.2 ± 0.2 pS with a linear current–voltage relationship. In CFTR+ cells, the whole-cell current at +90 mV increased from 7.3 ± 2.7 pA/pF (n = 12) to 46.1 ± 11.2 pA/pF (n = 5) after addition of dibutyryl-cyclic AMP (10−4 M) to the bath. Increasing the intracellular Cl− concentration to 150 mM activated linear Cl− currents in the absence of cyclic AMP in CFTR+ (n = 42) but not in CFTR− cells (n = 4). Similar Cl− current was also activated by high intracellular I− concentration. These results indicate that the CFTR-induced Cl− conductance in LTK− cells can be activated by either cyclic AMP or high intracellular halide concentrations.Key words: cystic fibrosis transmembrane conductance regulator (CFTR), chloride channel, cyclic AMP, whole-cell patch clamp, single-channel patch clamp.

A forskolin-activated Cl- current in mouse mandibular duct cells

1995 ◽  
Vol 268 (5) ◽  
pp. G806-G812 ◽  
Author(s):  
A. Dinudom ◽  
P. Komwatana ◽  
J. A. Young ◽  
D. I. Cook
Keyword(s):  
Mandibular Gland ◽  
Disulfonic Acid ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Beta Adrenergic ◽  
Beta Adrenergic Agonists ◽  
Duct Cells ◽  
Current Voltage ◽  
Cystic Fibrosis Transmembrane ◽  
Cl Conductance

We have previously shown that unstimulated granular duct cells of mouse mandibular gland contain a hyperpolarization-activated Cl- conductance with characteristics resembling the hyperpolarization-activated volume-sensitive Cl- channel (ClC-2). We now show that stimulation of these cells with forskolin, but not 1,9-dideoxyforskolin, activates a second whole cell Cl- conductance with properties resembling the cystic fibrosis transmembrane conductance regulator (CFTR). This conductance has a linear current-voltage relation and is not voltage activated. Its anion permeability sequence is Br- (1.96) > NO3- (1.36) > Cl- (1) > I- (0.44), and its conductance sequence is Cl- (1) > NO3- (0.66) > Br- (0.34) > I- (0.21). The current carried by this conductance is attenuated 65% by 1 mmol/l diphenylamine-2-carboxylate but is not affected by 0.1 mmol/l4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid or 0.1 mmol/l glibenclamide. The current can be activated by norepinephrine (1 mumol/l), evidently acting via beta-adrenergic receptors, since the effect of norepinephrine is inhibited by propranolol (1 mumol/l). We conclude that this adrenergically evoked conductance is due to CFTR, which has previously been shown to be expressed in salivary duct cells, and suggest that it may form part of the mechanism by which beta-adrenergic agonists modulate NaCl absorption by salivary ducts.

scholarly journals Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl- channel by two closely related arylaminobenzoates.

1993 ◽  
Vol 102 (1) ◽  
pp. 1-23 ◽  
Author(s):  
N A McCarty ◽  
S McDonough ◽  
B N Cohen ◽  
J R Riordan ◽  
N Davidson ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Binding Site ◽  
Open Channel ◽  
Transmembrane Conductance Regulator ◽  
Channel Block ◽  
Transmembrane Conductance ◽  
Open Channel Block ◽  
Whole Cell ◽  
Voltage Dependent ◽  
Cystic Fibrosis Transmembrane

The gene defective in cystic fibrosis encodes a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 microM, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses approximately 40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 +/- 0.4 pS in symmetric 150 mM Cl-. A subconductance state, measuring approximately 60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 microM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at Vm = -100 mV and not at Vm = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (approximately 1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at Vm = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway.

Sign in / Sign up

Export Citation Format

Share Document