Rectification of whole cell cystic fibrosis transmembrane conductance regulator chloride current

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.

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

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y93-094 ◽

1993 ◽

Vol 71 (9) ◽

pp. 645-649 ◽

Cited By ~ 5

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.

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Decreased expression of cystic fibrosis transmembrane conductance regulator impairs sperm quality in aged men

Reproduction ◽

10.1530/rep-13-0146 ◽

2013 ◽

Vol 146 (6) ◽

pp. 637-645 ◽

Cited By ~ 15

Author(s):

Ruiying Diao ◽

Kin Lam Fok ◽

Li Zhao ◽

Hao Chen ◽

Hui Tang ◽

...

Keyword(s):

Cystic Fibrosis ◽

Old Age ◽

Sperm Quality ◽

Neck Region ◽

Dependent Manner ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Age Dependent ◽

Old Men ◽

Cystic Fibrosis Transmembrane

Sperm quality declines with aging; however, the underlying molecular mechanism remains elusive. The cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to play an essential role in fertilizing capacity of sperm and male fertility. This study aimed to investigate the involvement of age-dependent CFTR downregulation in lowering sperm quality in old age. Two hundred and one healthy fertile men of three age groups (20–40 years,n=64; 40–60 years,n=61; and >60 years,n=76) were recruited. Expression of CFTR was determined by RT-PCR, western blot, and immunofluorescence staining. Collected sperm were treated with CFTR inhibitor or potentiator. Sperm quality was assessed by motility and bicarbonate-induced capacitation. The results showed that the expression of CFTR on the equatorial segment and neck region of sperm was significantly decreased in an age-dependent manner. Reduction of CFTR expression in sperm from old men was correlated with lowered forward motility and decreased HCO3−sensitivity required for sperm capacitation. Activation of CFTR by genistein partially rescued the decreased forward motility in sperm from old men. Decreased CFTR expression in sperm was also found to be associated with lowered sperm quality in aging mice. These results suggest that age-dependent downregulation of CFTR in sperm leads to lowered sperm quality in old age sperm. CFTR may be a pontential target for rescuing sperm motility as well as a fertility indicator in old age men.

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Fluoride stimulates cystic fibrosis transmembrane conductance regulator Cl− channel activity

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.274.3.l305 ◽

1998 ◽

Vol 274 (3) ◽

pp. L305-L312 ◽

Cited By ~ 1

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.

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

The Journal of General Physiology ◽

10.1085/jgp.102.1.1 ◽

1993 ◽

Vol 102 (1) ◽

pp. 1-23 ◽

Cited By ~ 110

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.

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