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.

scholarly journals The Pore Architecture of the Cystic Fibrosis Transmembrane Conductance Regulator Channel Revealed by Co-Mutation in Pore-Forming Transmembrane Regions

2016 ◽  
pp. 505-515
Author(s):  
F. QIAN ◽  
L. LIU ◽  
Z. LIU ◽  
C. LU
Keyword(s):  
Cystic Fibrosis ◽  
Single Channel ◽  
Transmembrane Conductance Regulator ◽  
Patch Clamp Recording ◽  
Transmembrane Conductance ◽  
Channel Pore ◽  
Selective Filter ◽  
Transmembrane Regions ◽  
Cystic Fibrosis Transmembrane ◽  
Insight Into

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel contains 12 transmembrane (TM) regions that are presumed to form the channel pore. However, there is no direct evidence clearly illustrating the involvement of these transmembrane regions in the actual CFTR pore structure. To obtain insight into the architecture of the CFTR channel pore, we used patch clamp recording techniques and a strategy of co-mutagenesis of two potential pore-forming transmembrane regions (TM1 and TM6) to investigate the collaboration of these two TM regions. We performed a range of specific functional assays comparing the single channel conductance, anion binding, and anion selectivity properties of the co-mutated CFTR variants, and the results indicated that TM1 and TM6 play vital roles in forming the channel pore and, thus, determine the functional properties of the channel. Furthermore, we provided functional evidence that the amino acid threonine (T338) in TM6 has synergic effects with lysine (K95) in TM1. Therefore, we propose that these two residues have functional collaboration in the CFTR channel pore and may collectively form a selective filter.

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.

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.

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.

Fluoride stimulates cystic fibrosis transmembrane conductance regulator Cl− channel activity

1998 ◽  
Vol 274 (3) ◽  
pp. L305-L312 ◽  
Author(s):  
Herbert A. Berger ◽  
Sue M. Travis ◽  
Michael J. Welsh
Keyword(s):  
Cystic Fibrosis ◽  
Single Channel ◽  
Single Channels ◽  
Dependent Manner ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
Cytosolic Domains ◽  
Cystic Fibrosis Transmembrane ◽  
Dose Dependent

While studying the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), we found that addition of F− to the cytosolic surface of excised, inside-out membrane patches reversibly increased Cl− current in a dose-dependent manner. Stimulation required prior phosphorylation and the presence of ATP. F− increased current even in the presence of deferoxamine, which chelates Al3+, suggesting that stimulation was not due to A[Formula: see text]. F− also stimulated current in a CFTR variant that lacked a large part of the R domain, suggesting that the effect was not mediated via this domain. Studies of single channels showed that F−increased the open-state probability by slowing channel closure from bursts of activity; the mean closed time between bursts and single-channel conductance was not altered. These results suggested that F− influenced regulation by the cytosolic domains, most likely the nucleotide-binding domains (NBDs). Consistent with this, we found that mutation of a conserved Walker lysine in NBD2 changed the relative stimulatory effect of F− compared with wild-type CFTR, whereas mutation of the Walker lysine in NBD1 had no effect. Based on these and previous data, we speculate that F− interacts with CFTR, possibly via NBD2, and slows the rate of channel closure.

scholarly journals Potentiation of the cystic fibrosis transmembrane conductance regulator Cl− channel by ivacaftor is temperature independent

2018 ◽  
Vol 315 (5) ◽  
pp. L846-L857 ◽  
Author(s):  
Yiting Wang ◽  
Zhiwei Cai ◽  
Martin Gosling ◽  
David N. Sheppard
Keyword(s):  
Cystic Fibrosis ◽  
Temperature Dependence ◽  
Single Channel ◽  
Channel Gating ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Cystic Fibrosis Transmembrane

Ivacaftor is the first drug to target directly defects in the cystic fibrosis transmembrane conductance regulator (CFTR), which causes cystic fibrosis (CF). To understand better how ivacaftor potentiates CFTR channel gating, here we investigated the effects of temperature on its action. As a control, we studied the benzimidazolone UCCF-853, which potentiates CFTR by a different mechanism. Using the patch-clamp technique and cells expressing recombinant CFTR, we studied the single-channel behavior of wild-type and F508del-CFTR, the most common CF mutation. Raising the temperature of the intracellular solution from 23 to 37°C increased the frequency but reduced the duration of wild-type and F508del-CFTR channel openings. Although the open probability ( Po) of wild-type CFTR increased progressively as temperature was elevated, the relationship between Po and temperature for F508del-CFTR was bell-shaped with a maximum Po at ~30°C. For wild-type CFTR and to a greatly reduced extent F508del-CFTR, the temperature dependence of channel gating was asymmetric with the opening rate demonstrating greater temperature sensitivity than the closing rate. At all temperatures tested, ivacaftor and UCCF-853 potentiated wild-type and F508del-CFTR. Strikingly, ivacaftor but not UCCF-853 abolished the asymmetric temperature dependence of CFTR channel gating. At all temperatures tested, Po values of wild-type CFTR in the presence of ivacaftor were approximately double those of F508del-CFTR, which were equivalent to or greater than those of wild-type CFTR at 37°C in the absence of the drug. We conclude that the principal effect of ivacaftor is to promote channel opening to abolish the temperature dependence of CFTR channel gating.

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