CFTR: Ligand exchange between a permeant anion ([Au(CN)2]-) and an engineered cysteine (T338C) blocks the pore

Previous attempts to identify residues that line the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have utilized cysteine-substituted channels in conjunction with impermeant, thiol-reactive reagents like MTSET+ and MTSES-. We report here that the permeant, pseudohalide anion [Au(CN)2]- can also react with a cysteine engineered into the pore of the CFTR channel. Exposure of Xenopus oocytes expressing the T338C CFTR channel to as little as 100 nM [Au(CN)2]- produced a profound reduction in conductance that was not reversed by washing but was reversed by exposing the oocytes to a competing thiol like DTT (dithiothreitol) and 2-ME (2-mercaptoethanol). In detached, inside out patches single-channel currents were abolished by [Au(CN)2]- and activity was not restored by washing [Au(CN)2]- from the bath. Both single-channel and macroscopic currents were restored, however, by exposing [Au(CN)2]- -blocked channels to excess [CN]-. The results are consistent with the hypothesis that [Au(CN)2]- can participate in a ligand exchange reaction with the cysteine thiolate at 338 such that the mixed-ligand complex, with a charge of -1, blocks the anion conduction pathway.     

CFTR: covalent modification of cysteine-substituted channels expressed in Xenopus oocytes shows that activation is due to the opening of channels resident in the plasma membrane

Some studies of CFTR imply that channel activation can be explained by an increase in open probability (P(o)), whereas others suggest that activation involves an increase in the number of CFTR channels (N) in the plasma membrane. Using two-electrode voltage clamp, we tested for changes in N associated with activation of CFTR in Xenopus oocytes using a cysteine-substituted construct (R334C CFTR) that can be modified by externally applied, impermeant thiol reagents like [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET+). Covalent modification of R334C CFTR with MTSET+ doubled the conductance and changed the I-V relation from inward rectifying to linear and was completely reversed by 2-mercaptoethanol (2-ME). Thus, labeled and unlabeled channels could be differentiated by noting the percent decrease in conductance brought about by exposure to 2-ME. When oocytes were briefly (20 s) exposed to MTSET+ before CFTR activation, the subsequently activated conductance was characteristic of labeled R334C CFTR, indicating that the entire pool of CFTR channels activated by cAMP was accessible to MTSET+. The addition of unlabeled, newly synthesized channels to the plasma membrane could be monitored on-line during the time when the rate of addition was most rapid after cRNA injection. The addition of new channels could be detected as early as 5 h after cRNA injection, occurred with a half time of approximately 24-48 h, and was disrupted by exposing oocytes to Brefeldin A, whereas activation of R334C CFTR by cAMP occurred with a half time of tens of minutes, and did not appear to involve the addition of new channels to the plasma membrane. These findings demonstrate that in Xenopus oocytes, the major mechanism of CFTR activation by cAMP is by means of an increase in the open probability of CFTR channels.     

Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride

In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action.     

Variable reactivity of an engineered cysteine at position 338 in cystic fibrosis transmembrane conductance regulator reflects different chemical states of the thiol

In a previous study of T338C CFTR (cystic fibrosis transmembrane conductance regulator) we found that protons and thiol-directed reagents modified channel properties in a manner consistent with the hypothesis that this residue lies within the conduction path, but the observed reactivity was not consistent with the presence of a single thiolate species in the pore. Here we report results consistent with the notion that the thiol moiety can exist in at least three chemical states, the simple thiol, and two altered states. One of the altered states displays reactivity toward thiols like dithiothreitol and 2-mercaptoethanol as well as reagents: mixed disulfides (methanethiosulfonate reagents: MTSET+, MTSES-) and an alkylating agent (iodoacetamide). The other altered state is unreactive. The phenotype associated with the reactive, altered state could be replicated by exposing oocytes expressing T338C CFTR to CuCl2, but not by glutathionylation or nitrosylation of the thiol or by oxidation with hydrogen peroxide. The results are consistent with the hypothesis that substituting a cysteine at 338 can create an adventitious metal binding site. Metal liganding alters thiol reactivity and may, in some cases, catalyze oxidation of the thiol to an unreactive form such as a sulfinic or sulfonic acid.     

CFTR: a cysteine at position 338 in TM6 senses a positive electrostatic potential in the pore

We investigated the accessibility to protons and thiol-directed reagents of a cysteine substituted at position 338 in transmembrane segment 6 (TM6) of CFTR to test the hypothesis that T338 resides in the pore. Xenopus oocytes expressing T338C CFTR exhibited pH-dependent changes in gCl and I-V shape that were specific to the substituted cysteine. The apparent pKa of T338C CFTR was more acidic than that expected for a cysteine or similar simple thiols in aqueous solution. The pKa was shifted toward alkaline values when a nearby positive charge (R334) was substituted with neutral or negatively charged residues, consistent with the predicted influence of the positive charge of R334, and perhaps other residues, on the titration of a cysteine at 338. The relative rates of chemical modification of T338C CFTR by MTSET+ and MTSES- were also altered by the charge at 334. These observations support a model for CFTR that places T338 within the anion conduction path. The apparent pKa of a cysteine substituted at 338 and the relative rates of reaction of charged thiol-directed reagents provide a crude measure of a positive electrostatic potential that may be due to R334 and other residues near this position in the pore.     

Mercury and zinc differentially inhibit shark and human CFTR orthologues: involvement of shark cysteine 102

The apical membrane is an important site of mercury toxicity in shark rectal gland tubular cells. We compared the effects of mercury and other thiol-reacting agents on shark CFTR (sCFTR) and human CFTR (hCFTR) chloride channels using two-electrode voltage clamping of cRNA microinjected Xenopus laevis oocytes. Chloride conductance was stimulated by perfusing with 10 µM forskolin (FOR) and 1 mM IBMX, and then thio-reactive species were added. In oocytes expressing sCFTR, FOR + IBMX mean stimulated Cl^– conductance was inhibited 69% by 1 µM mercuric chloride and 78% by 5 µM mercuric chloride (IC₅₀ of 0.8 µM). Despite comparable stimulation of conductance, hCFTR was insensitive to 1 µM HgCl₂ and maximum inhibition was 15% at the highest concentration used (5 µM). Subsequent exposure to glutathione (GSH) did not reverse the inhibition of sCFTR by mercury, but dithiothreitol (DTT) completely reversed this inhibition. Zinc (50–200 µM) also reversibly inhibited sCFTR (40–75%) but did not significantly inhibit hCFTR. Similar inhibition of sCFTR but not hCFTR was observed with an organic mercurial, p-chloromercuriphenylsulfonic acid (pCMBS). The first membrane spanning domain (MSD1) of sCFTR contains two unique cysteines, C102 and C303. A chimeric construct replacing MSD1 of hCFTR with the corresponding sequence of sCFTR was highly sensitive to mercury. Site-specific mutations introducing the first but not the second shark unique cysteine in hCFTR MSD1 resulted in full sensitivity to mercury. These experiments demonstrate a profound difference in the sensitivity of shark vs. human CFTR to inhibition by three thiol-reactive substances, an effect that involves C102 in the shark orthologue. chloride transport; Xenopus laevis oocytes; dithiothreitol; glutathione; p-chloromercuriphenylsulfonic acid; cystic fibrosis transmembrane regulator     

Redox-dependent modulation of the carrot SV channel by cytosolic pH

Currents mediated by a slow vacuolar (SV) channel were recorded and characterized in vacuoles from cultured carrot cells. The carrot channel shows the typical functional characteristics reported for channels of the SV category previously identified in other plants, i.e., slow voltage-dependent activation kinetics, current activation favoured by cytosolic calcium and permeability to different monovalent cations. The carrot channel is strongly activated by cytosolic reducing agents (such as dithiothreitol, DTT, and glutathione, GSH) and has a peculiar dependence on cytosolic pH, which, in turn, is affected by the concentration of cytosolic reducing agents. Specifically, in 1 mM DTT or GSH the channel displayed a maximum conductance at neutral pH. The normalized conductance did not depend significantly on DTT concentration at acidic pH, while at alkaline pH the attenuation of the normalized conductance declines with increasing DTT concentration. Our results suggest two pH-titratable groups within the carrot SV channel, one of these depending on cysteine residues exposed to the cytosolic side of the vacuole.     

Glutathione levels and BAX activation during apoptosis due to oxidative stress in cells expressing wild-type and mutant cystic fibrosis transmembrane conductance regulator

Cystic fibrosis is characterized by chronic inflammation and an imbalance in the concentrations of alveolar and lung oxidants and antioxidants, which result in cell damage. Modifications in lung glutathione concentrations are recognized as a salient feature of inflammatory lung diseases such as cystic fibrosis, and glutathione plays a major role in protection against oxidative stress and is important in modulation of apoptosis. The cystic fibrosis transmembrane conductance regulator (CFTR) is permeable to Cl(-), larger organic ions, and reduced and oxidized forms of glutathione, and the DeltaF508 CFTR mutation found in cystic fibrosis patients has been correlated with impaired glutathione transport in cystic fibrosis airway epithelia. Because intracellular glutathione protects against oxidative stress-induced apoptosis, we studied the susceptibility of epithelial cells (HeLa and IB3-1) expressing normal and mutant CFTR to apoptosis triggered by H(2)O(2). We find that cells with normal CFTR are more sensitive to oxidative stress-induced apoptosis than cells expressing defective CFTR. In addition, sensitivity to apoptosis could be correlated with glutathione levels, because depletion of intracellular glutathione results in higher levels of apoptosis, and glutathione levels decreased faster in cells expressing normal CFTR than in cells with defective CFTR during incubation with H(2)O(2). The pro-apoptotic BCL-2 family member, BAX, is also activated faster in cells expressing normal CFTR than in those with mutant CFTR under these conditions, and artificial glutathione depletion increases the extent of BAX activation. These results suggest that glutathione-dependent BAX activation in cells with normal CFTR represents an early step in oxidative stress-induced apoptosis of these cells.     

The CLIC1 Chloride Channel Is Regulated by the Cystic Fibrosis Transmembrane Conductance Regulator when Expressed in Xenopus Oocytes

CLIC proteins comprise a family of chloride channels whose physiological roles are uncertain. To gain further insight into possible means of CLIC1 channel activity regulation, this protein was expressed in Xenopus oocytes alone or in combination with the cystic fibrosis transmembrane conductance regulator (CFTR). Whole-cell currents were determined using two-electrode voltage-clamp methods. Expression of CLIC1 alone did not increase whole-cell conductance either at rest or in response to increased intracellular cyclic adenosine monophosphate (cAMP). However, expression of CLIC1 with CFTR led to increased cAMP-activated whole-cell currents compared to expression from the same amount of CFTR mRNA alone. IAA-94 is a drug known to inhibit CLIC family channels but not CFTR. In oocytes expressing both CLIC1 and CFTR, a fraction of the cAMP-activated whole-cell current was sensitive to IAA-94, whereas in oocytes expressing CFTR alone, the cAMP-stimulated current was resistant to the drug. Cell fractionation studies revealed that the presence of CFTR conferred cAMP-stimulated redistribution of a fraction of CLIC1 from a soluble to a membrane-associated form. We conclude that when expressed in Xenopus oocytes CFTR confers cAMP regulation to CLIC1 activity in the plasma membrane and that at least part of this regulation is due to recruitment of CLIC1 from the cytoplasm to the membrane.     

Functional characterization of a novel CFTR mutation P67S identified in a patient with atypical cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR serves as a cAMP-stimulated chloride channel in a wide range of epithelial tissues and its dysfunction is a hallmark of CF. Over 1400 mutations in the CFTR gene are known, but functional data exist only for a minority of the mutant channels. The aim of the present study was to functionally characterize a novel CFTR mutation identified in a patient with atypical CF. Full length sequencing of the patient's CFTR gene revealed a homozygous C to T transition at nucleotide position 331 (CCT>TCT), which results in a P67S amino acid substitution. Mutant and wild-type CFTR were heterologously expressed in Xenopus laevis oocytes. CFTR whole-cell currents were studied using the two-electrode voltage-clamp technique. Channel surface expression was assessed by a chemiluminescence assay. Expression of P67S-CFTR resulted in functional CFTR chloride channels. However, the CFTR chloride conductance observed in oocytes expressing the mutant channel averaged only 24% of that in oocytes expressing wild-type CFTR. Similarly, surface expression of the mutant channel was reduced. In contrast, the mutation did not alter the anion selectivity of the channel, and Western blot analysis indicated a similar protein expression level of mutant and wild-type CFTR. Our findings indicate that the P67S mutation reduces CFTR chloride channel function by reducing channel surface expression. The mild disease phenotype of the patient indicates that the residual function of the mutant channel is sufficient to prevent the development of severe CF symptoms.     

Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C

We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.     

Thermal instability of ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) channel function: protection by single suppressor mutations and inhibiting channel activity

Deletion of Phe508 from cystic fibrosis transmembrane conductance regulator (CFTR) results in a temperature-sensitive folding defect that impairs protein maturation and chloride channel function. Both of these adverse effects, however, can be mitigated to varying extents by second-site suppressor mutations. To better understand the impact of second-site mutations on channel function, we compared the thermal sensitivity of CFTR channels in Xenopus oocytes. CFTR-mediated conductance of oocytes expressing wt or ΔF508 CFTR was stable at 22 °C and increased at 28 °C, a temperature permissive for ΔF508 CFTR expression in mammalian cells. At 37 °C, however, CFTR-mediated conductance was further enhanced, whereas that due to ΔF508 CFTR channels decreased rapidly toward background, a phenomenon referred to here as "thermal inactivation." Thermal inactivation of ΔF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K, and R1070W, but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Another mutation, K1250A, known to increase open probability (P(o)) of ΔF508 CFTR channels, exacerbated thermal inactivation. Application of potentiators known to increase P(o) of ΔF508 CFTR channels at room temperature failed to protect channels from inactivation at 37 °C and one, PG-01, actually exacerbated thermal inactivation. Unstimulated ΔF508CFTR channels or those inhibited by CFTR(inh)-172 were partially protected from thermal inactivation, suggesting a possible inverse relationship between thermal stability and gating transitions. Thermal stability of channel function and temperature-sensitive maturation of the mutant protein appear to reflect related, but distinct facets of the ΔF508 CFTR conformational defect, both of which must be addressed by effective therapeutic modalities.     

Asymmetry in the Osmotic Response of a Rat Cortical Collecting Duct Cell Line: Role of Aquaporin-2

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl⁻) channel known to influence the function of other channels, including connexin channels. To further study potential functional interactions between CFTR and gap junction channels, we have co-expressed CFTR and connexin45 (Cx45) in Xenopus oocytes and monitored junctional conductance and voltage sensitivity by dual voltage clamp electrophysiology. In single oocytes expressing CFTR, an increase in cAMP caused by forskolin application induced a Cl⁻ current and increased membrane conductance; application of diphenylamine carboxylic acid (CFTR blocker) readily blocked the Cl⁻ current. With co-expression of CFTR and Cx45, application of forskolin to paired oocytes induced a typical outward current and increased junctional conductance (G_j). In addition, the presence of CFTR reduced the transjunctional voltage sensitivity of Cx45 channels without affecting the kinetics of junctional current inactivation. The drop in voltage sensitivity was further enhanced by forskolin application. The data indicate that CFTR influences cell-to-cell coupling mediated by Cx45 channels.     

Regulation of CFTR trafficking by its R domain

Phosphorylation of the R domain is required for cystic fibrosis transmembrane conductance regulator (CFTR) channel gating, and cAMP/protein kinase A (PKA) simulation can also elicit insertion of CFTR into the plasma membrane from intracellular compartments (Bertrand, C. A., and Frizzell, R. A. (2003) Am. J. Physiol. 285, C1-C18). We evaluated the structural basis of regulated CFTR trafficking by determining agonist-evoked increases in plasma membrane capacitance (Cm) of Xenopus oocytes expressing CFTR deletion mutants. Expression of CFTR as a split construct that omitted the R domain (Deltaamino acids 635-834) produced a channel with elevated basal current (Im) and no DeltaIm or trafficking response (DeltaCm) upon cAMP/PKA stimulation, indicating that the structure(s) required for regulated CFTR trafficking are contained within the R domain. Additional deletions showed that removal of amino acids 817-838, a 22-amino acid conserved helical region having a net charge of -9, termed NEG2 (Xie, J., Adams, L. M., Zhao, J., Gerken, T. A., Davis, P. B., and Ma, J. (2002) J. Biol. Chem. 277, 23019-23027), produced a channel with regulated gating that lacked the agonist-induced increase in CFTR trafficking. Injection of NEG2 peptides into oocytes expressing split DeltaNEG2 CFTR prior to stimulation restored the agonist-evoked DeltaCm, consistent with the concept that this sequence mediates the regulated trafficking event. In support of this idea, DeltaNEG2 CFTR escaped from the inhibition of wild type CFTR trafficking produced by overexpression of syntaxin 1A. These observations suggest that the NEG2 region at the C terminus of the R domain allows stabilization of CFTR in a regulated intracellular compartment from which it traffics to the plasma membrane in response to cAMP/PKA stimulation.     

Ca(2+)-activated K+ channel inhibition by reactive oxygen species

We studied the effect of H(2)O(2) on the gating behavior of large-conductance Ca(2+)-sensitive voltage-dependent K(+) (K(V,Ca)) channels. We recorded potassium currents from single skeletal muscle channels incorporated into bilayers or using macropatches of Xenopus laevis oocytes membranes expressing the human Slowpoke (hSlo) alpha-subunit. Exposure of the intracellular side of K(V,Ca) channels to H(2)O(2) (4-23 mM) leads to a time-dependent decrease of the open probability (P(o)) without affecting the unitary conductance. H(2)O(2) did not affect channel activity when added to the extracellular side. These results provide evidence for an intracellular site(s) of H(2)O(2) action. Desferrioxamine (60 microM) and cysteine (1 mM) completely inhibited the effect of H(2)O(2), indicating that the decrease in P(o) was mediated by hydroxyl radicals. The reducing agent dithiothreitol (DTT) could not fully reverse the effect of H(2)O(2). However, DTT did completely reverse the decrease in P(o) induced by the oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid). The incomplete recovery of K(V,Ca) channel activity promoted by DTT suggests that H(2)O(2) treatment must be modifying other amino acid residues, e.g., as methionine or tryptophan, besides cysteine. Noise analysis of macroscopic currents in Xenopus oocytes expressing hSlo channels showed that H(2)O(2) induced a decrease in current mediated by a decrease both in the number of active channels and P(o).     

Epithelial sodium channels regulate cystic fibrosis transmembrane conductance regulator chloride channels in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl(-) channel properties, regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and nonepithelial cells. Because modulation of net NaCl reabsorption has important implications in extracellular fluid volume homeostasis and airway fluid volume and composition, we investigated whether this regulation was reciprocal by examining whether ENaC regulates CFTR. Co-expression of human (h) CFTR and mouse (m) alphabetagammaENaC in Xenopus oocytes resulted in a significant, 3.7-fold increase in whole-cell hCFTR Cl(-) conductance compared with oocytes expressing hCFTR alone. The forskolin/3-isobutyl-1-methylxanthine-stimulated whole-cell conductance in hCFTR-mENaC co-injected oocytes was amiloride-insensitive, indicating an inhibition of mENaC following hCFTR activation, and it was blocked by DPC (diphenylamine-2-carboxylic acid) and was DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid)-insensitive. Enhanced hCFTR Cl(-) conductance was also observed when either the alpha- or beta-subunit of mENaC was co-expressed with hCFTR, but this was not seen when CFTR was co-expressed with the gamma-subunit of mENaC. Single Cl(-) channel analyses showed that both CFTR Cl(-) channel open probability and the number of CFTR Cl(-) channels detected per patch increased when hCFTR was co-expressed with alphabetagammamENaC. We conclude that in addition to acting as a regulator of ENaC, CFTR activity is regulated by ENaC.     

Enhancement of intracellular glutathione promotes lymphocyte activation by mitogen

We have examined the effect of chemically modulating intracellular glutathione (GSH) levels on murine lymphocyte activation. Lymphocyte activation was determined by the induction of polyamine synthesis (ornithine decarboxylase (ODC) induction) and DNA synthesis ([3H]thymidine([3H]Tdr) incorporation). Intracellular GSH levels were enhanced using L-2-oxothiazolidine-4-carboxylate (OTC), which delivers cysteine intracellularly, and suppressed by buthionine sulfoximine (BSO), which inhibits gamma-glutamylcysteine synthetase. In addition, the thiol 2-mercaptoethanol (2-ME) was tested for its ability to augment intracellular GSH levels. Our results indicate that both OTC and 2-ME enhance GSH concentrations and [3H]Tdr incorporation in resting and mitogen (concanavalin A)-stimulated cells. The induction of ODC by concanavalin A (Con A) was augmented by the addition of OTC or 2-ME. The GSH concentration of Con A-stimulated cells was reduced when compared to resting cells; however, it was markedly enhanced by OTC or 2-ME. The stimulatory effects of 2-ME on GSH concentrations, [3H]Tdr incorporation, and ODC induction in both resting and Con A-stimulated cells were much more potent than those of OTC. In contrast, BSO suppressed intracellular GSH and [3H]Tdr incorporation in resting and Con A-stimulated cells. BSO also inhibited the promotion of intracellular GSH concentrations and [3H]Tdr uptake by OTC or 2-ME. However, BSO did not affect the induction of ODC by Con A or its enhancement by OTC or 2-ME. We conclude that enhancement of intracellular GSH concentration results in an increased lymphocyte response to mitogen stimulation.     

Reversible silencing of CFTR chloride channels by glutathionylation

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and ATP-dependent chloride channel that modulates salt and water transport across lung and gut epithelia. The relationship between CFTR and oxidized forms of glutathione is of potential interest because reactive glutathione species are produced in inflamed epithelia where they may be modulators or substrates of CFTR. Here we show that CFTR channel activity in excised membrane patches is markedly inhibited by several oxidized forms of glutathione (i.e., GSSG, GSNO, and glutathione treated with diamide, a strong thiol oxidizer). Three lines of evidence indicate that the likely mechanism for this inhibitory effect is glutathionylation of a CFTR cysteine (i.e., formation of a mixed disulfide with glutathione): (a) channels could be protected from inhibition by pretreating the patch with NEM (a thiol alkylating agent) or by lowering the bath pH; (b) inhibited channels could be rescued by reducing agents (e.g., DTT) or by purified glutaredoxins (Grxs; thiol disulfide oxidoreductases) including a mutant Grx that specifically reduces mixed disulfides between glutathione and cysteines within proteins; and (c) reversible glutathionylation of CFTR polypeptides in microsomes could be detected biochemically under the same conditions. At the single channel level, the primary effect of reactive glutathione species was to markedly inhibit the opening rates of individual CFTR channels. CFTR channel inhibition was not obviously dependent on phosphorylation state but was markedly slowed when channels were first "locked open" by a poorly hydrolyzable ATP analogue (AMP-PNP). Consistent with the latter finding, we show that the major site of inhibition is cys-1344, a poorly conserved cysteine that lies proximal to the signature sequence in the second nucleotide binding domain (NBD2) of human CFTR. This region is predicted to participate in ATP-dependent channel opening and to be occluded in the nucleotide-bound state of the channel based on structural comparisons to related ATP binding cassette transporters. Our results demonstrate that human CFTR channels are reversibly inhibited by reactive glutathione species, and support an important role of the region proximal to the NBD2 signature sequence in ATP-dependent channel opening.     

Slow conversions among subconductance states of cystic fibrosis transmembrane conductance regulator chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel exhibits multiple subconductance states. To study the regulation of conductance states of the CFTR channel, we expressed the wild-type CFTR protein in HEK 293 cells, and isolated microsomal membrane vesicles for reconstitution studies in lipid bilayer membranes. A single CFTR channel had a dominant conductance of 7.8 pS (H), plus two sub-open states with conductances of approximately 6 pS (M) and 2.7 pS (L) in 200 mM KCl with 1 mM MgCl2 (intracellular) and 50 mM KCl with no MgCl2 (extracellular), with pH maintained at 7.4 by 10 mM HEPES-Tris on both sides of the channel. In 200 mM KCl, both H and L states could be measured in stable single-channel recordings, whereas M could not. Spontaneous transitions between H and L were slow; it took 4.5 min for L-->H, and 3.2 min for H-->L. These slow conversions among subconductance states of the CFTR channel were affected by extracellular Mg; in the presence of millimolar Mg, the channel remained stable in the H state. Similar phenomena were also observed with endogenous CFTR channels in T84 cells. In high-salt conditions (1.5 M KCl), all three conductance states of the expressed CFTR channel, 12.1 pS, 8.2 pS, and 3.6 pS, became stable and seemed to gate independently from each other. The existence of multiple stable conductance states associated with the CFTR channel suggests two possibilities: either a single CFTR molecule can exist in multiple configurations with different conductance values, or the CFTR channel may contain multimers of the 170-kDa CFTR protein, and different conductance states are due to different aggregation states of the CFTR protein.     

Lubiprostone activates CFTR, but not ClC-2, via the prostaglandin receptor (EP4)

The goal of this study was to determine the mechanism of lubiprostone activation of epithelial chloride transport. Lubiprostone is a bicyclic fatty acid approved for the treatment of constipation [1]. There is uncertainty, however, as to how lubiprostone increases epithelial chloride transport. Direct stimulation of ClC-2 and CFTR chloride channels as well as stimulation of these channels via the EP₄ receptor has been described [2], [3], [4] and [5]. To better define this mechanism, two-electrode voltage clamp was used to assay Xenopus oocytes expressing ClC-2, with or without co-expression of the EP₄ receptor or β adrenergic receptor (βAR), for changes in conductance elicited by lubiprostone. Oocytes co-expressing CFTR and either βAR or the EP₄ receptor were also studied. In oocytes co-expressing ClC-2 and βAR conductance was stimulated by hyperpolarization and acidic pH (pH = 6), but there was no response to the β adrenergic agonist, isoproterenol. Oocytes expressing ClC-2 only or co-expressing ClC-2 and EP₄ did not respond to the presence of 0.1, 1, or 10 μM lubiprostone in the superperfusate. Oocytes co-expressing CFTR and βAR did not respond to hyperpolarization, acidic pH, or 1 μM lubiprostone. However, conductance was elevated by isoproterenol and inhibited by CFTR_inh172. Co-expression of CFTR and EP₄ resulted in lubiprostone-stimulated conductance, which was also sensitive to CFTR_inh172. The EC₅₀ for lubiprostone mediated CFTR activation was ∼10 nM. These results demonstrate no direct action of lubiprostone on either ClC-2 or CFTR channels expressed in oocytes. However, the results confirm that CFTR can be activated by lubiprostone via the EP₄ receptor in oocytes.