Outwardly Rectifying Cl- Channels Research Articles

Ca 2+-independent effects of BAPTA and EGTA on single-channel Cl − currents in brown adipocytes

The Cl − channels of brown adipocytes electrophysiologically resemble outwardly rectifying Cl − channels (ORCC). To study tentative Ca 2+ regulation of these channels, we attempted to control Ca 2+ levels at the cytoplasmic side of the inside-out membrane patches with Ca 2+-chelating agents. However, we found that the commonly used Ca 2+-chelators EGTA and BAPTA by themselves influenced the Cl − channel currents, unrelated to their calcium chelating effects. Consequently, in this report we delineate effects of Ca 2+-chelators (acting from the cytoplasmic side) on the single Cl − channel currents in patch-clamp experiments. Using fixed (1–2 mM) concentrations of chelators, two types of Cl − channels were identified, as discriminated by their reaction to the Ca 2+-chelators and by their conductance: true-blockage channels (31 pS) and quasi-blockage channels (52 pS). In true-blockage channels, EGTA and BAPTA inhibited channel activity in a classical flickery type manner. In quasi-blockage channels, chelators significantly shortened the duration of individual openings, as in a flickering block, but the overall channel activity tended to increase. This dual effect of mean open time decrease accompanied by a tendency of open probability to increase we termed a quasi-blockage. Despite the complications due to the chelators as such, we could detect a moderate inhibitory effect of Ca 2+. The anionic classical Cl − channel blockers DIDS and SITS could mimic the true/quasi blockage of EGTA and BAPTA. It was concluded that at least in this experimental system, standard techniques for Ca 2+ level control in themselves could fundamentally affect the behaviour of Cl − channels.

The Formation of the cAMP/Protein Kinase A-dependent Annexin 2–S100A10 Complex with Cystic Fibrosis Conductance Regulator Protein (CFTR) Regulates CFTR Channel Function

Cystic fibrosis results from mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/protein kinase A (PKA) and ATP-regulated Cl(-) channel. CFTR is increasingly recognized as a component of multiprotein complexes and although several inhibitory proteins to CFTR have been identified, protein complexes that stimulate CFTR function remain less well characterized. We report that annexin 2 (anx 2)-S100A10 forms a functional cAMP/PKA/calcineurin (CaN)-dependent complex with CFTR. Cell stimulation with forskolin/3-isobutyl-1-methylxanthine significantly increases the amount of anx 2-S100A10 that reciprocally coimmunoprecipitates with cell surface CFTR and calyculin A. Preinhibition with PKA or CaN inhibitors attenuates the interaction. Furthermore, we find that the acetylated peptide (STVHEILCKLSLEG, Ac1-14), but not the nonacetylated equivalent N1-14, corresponding to the S100A10 binding site on anx 2, disrupts the anx 2-S100A10/CFTR complex. Analysis of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and CFTR(inh172)-sensitive currents, taken as indication of the outwardly rectifying Cl(-) channels (ORCC) and CFTR-mediated currents, respectively, showed that Ac1-14, but not N1-14, inhibits both the cAMP/PKA-dependent ORCC and CFTR activities. CaN inhibitors (cypermethrin, cyclosporin A) discriminated between ORCC/CFTR by inhibiting the CFTR(inh172)-, but not the DIDS-sensitive currents, by >70%. Furthermore, peptide Ac1-14 inhibited acetylcholine-induced short-circuit current measured across a sheet of intact intestinal biopsy. Our data suggests that the anx 2-S100A10/CFTR complex is important for CFTR function across epithelia.

Transport of nutrients and electrolytes across the intestinal wall in pigs

Abstract In recent years, intestinal transport processes have been studied in detail regarding both, functional and structural aspects. For monosaccharides different systems have been demonstrated for apical uptake: this includes the high-affinity SGLT1 as a distinct d -glucose system and GLUT5 for fructose. Specifically in pigs a low affinity, high-capacity system for d -glucose and d -mannose with no preference for Na+ over K+ and a very low affinity system are suggested as further uptake systems. As in other species, basolateral extrusion is mediated by GLUT2. The distributions of monosaccharide transport along the gastrointestinal axis as well as the potential role of paracellular monosaccharide absorption have not yet been clarified. Amino acids can principally be absorbed by the paracellular and transcellular pathway whereas transcellular transport can either be mediated by facilitated diffusion or secondary active Na+-coupled transport. This includes different transport systems for neutral, anionic and cationic acids. In addition, the presence of the di-/tripeptides transport system PEPT1 which depends on an inwardly directed H+-gradient has also been confirmed for the pig small intestine, its quantitative proportion is still under debate. Short chain fatty acids (SCFA) are the major end products of microbial carbohydrate fermentation which occurs along the gastrointestinal tract with the highest production rates in the large intestines. At least two uptake mechanisms have to be assumed, i.e., non-ionic diffusion and anionic exchange via SCFA−/HCO3−-exchange. Controversial views still exist to what extent SCFA are metabolized within the epithelial tissue. Segmental differences between small and large intestines have been demonstrated for Na+ absorption. Whereas in the small intestines the major part of Na+ absorption is mediated by coupled nutrient transport systems, aldosterone sensitive Na+ channels and Na+/H+-exchange are the dominant mechanisms in the hindgut. For Cl− paracellular transport and anionic Cl−/HCO3−-exchange are the major absorptive mechanisms. Cl− secretion is mediated by apical channels which may be activated by toxins of different origin. Different types of Cl− channels have been identified, such as Cystic Fibrosis Transmembrane Regulator (CFTR), Ca-activated Cl− channels (CLCA) and Outwardly Rectifying Cl− Channels (ORCC). Whereas CFTR has clearly been shown for jejunal and colonic epithelial and goblet cells controversy still exists on the relevance of CLCA and ORCC in pigs. For Ca2+ there is evidence that both recently published channels TRPV5 and TRPV6 are also expressed in pig intestinal tissues, however, this has not yet been shown on protein level. From several functional approaches it was demonstrated that phosphate uptake can be mediated by both, a Na+-dependent transcellular component and paracellularly. On a molecular basis it is uncertain whether the transport protein of transcellular mechanism belongs to the NaPi-IIb cotransporter family.

Structure and function of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR). Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel. Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients. The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP. The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs), and a cytoplasmic domain. In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Cl- channels (ORCCs).

Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator.

CFTR is a cyclic AMP (cAMP)-activated chloride (Cl-) channel and a regulator of outwardly rectifying Cl- channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl- channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3-1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36Cl- efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl- channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted alpha-helices 5 and 6, forms an essential part of the Cl- channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl- channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.

Identification of a New Outwardly Rectifying Cl− Channel That Belongs to a Subfamily of the ClC Cl− Channels

A new outwardly rectifying Cl- channels (ORCC) that belongs to ClC Cl- channel family has been identified from rat kidney and designated as ClC-5. ClC-5 cDNA encodes a polypeptide of 746 amino acids, which is indicated by hydrophobicity analysis to have structural features that are common of the ClC family. However, the amino acid sequence was weakly homologous to those of other ClC Cl- channels except for ClC-3, which we recently identified as a Ca2+-sensitive ORCC. Northern blot analysis of rat tissues showed that ClC-5 mRNA was predominantly expressed in the kidney and colon. To characterize the functional properties of ClC-5 by whole cell patch-clamp technique, we established the stably transfected CHO-K1 cell line using intranuclear microinjection technique. The transfected cells induced outwardly rectifying and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive Cl- currents on whole cell configuration. Following the identification of two highly homologous ORCCs, ClC-3 and ClC-5, a new subfamily encoding ORCC has emerged in the ClC family. Furthermore, ClC-5 was almost identical to a partial sequence of human cDNA that is related to Dent's disease. The molecular structure and functional properties of ClC-5 will provide an important insight into ORCCs and the pathogenesis of Dent's disease.

G protein G alpha i-2 inhibits outwardly rectifying chloride channels in 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.

CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP

The cystic fibrosis transmembrane conductance regulator (CFTR) functions to regulate both Cl- and Na+ conductive pathways; however, the cellular mechanisms whereby CFTR acts as a conductance regulator are unknown. CFTR and outwardly rectifying Cl- channels (ORCCs) are distinct channels but are linked functionally via an unknown regulatory mechanism. We present results from whole-cell and single-channel patch-clamp recordings, short-circuit current recordings, and [gamma-32P]ATP release assays of normal, CF, and wild-type or mutant CFTR-transfected CF airway cultured epithelial cells wherein CFTR regulates ORCCs by triggering the transport of the potent agonist, ATP, out of the cell. Once released, ATP stimulates ORCCs through a P2U purinergic receptor-dependent signaling mechanism. Our results suggest that CFTR functions to regulate other Cl- secretory pathways in addition to itself conducting Cl-.

Protein kinase A-regulated Cl- channel in ML-1 human hematopoietic myeloblasts.

Using the inside-out patch clamp technique, we identified a Cl- channel in patches from the membrane of cultured human hematopoietic myeloblastic leukemia ML-1 cells. The Cl- channel was not seen at negative membrane potentials in excised patches until the membrane potential was depolarized to greater than +40 mV. The channel was also activated by addition of cAMP-dependent protein kinase (PKA) catalytic subunit at physiological membrane potential (-40 mV). Biophysical studies of the Cl- channel revealed that the current-voltage (I-V) relationship of the Cl- channel was outwardly rectifying in symmetrical 142 mM Cl- solutions. Single channel conductances were 48 pS for the outward current measured at +60 mV and 27 pS for the inward current at -60 mV. The open time constant of the channel was dependent on the membrane potential and was significantly prolonged at positive membrane potentials. Channels activated by cAMP-dependent protein kinase spent a significantly longer time in the open state compared to those channels activated by depolarization pulses. Pharmacological properties of the Cl- channel were also studied. Two anion transport inhibitors, anthracene-9-carboxylic acid (9-AC) and 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) caused a flickering block of the channel. Half-inhibitory concentrations (IC50) for 9-AC and DIDS were 174 +/- 20 and 70 +/- 16 microM, respectively. Blockade of the Cl- channel by 9-AC or DIDS was completely reversible. Our findings suggest that outwardly rectifying Cl- channels (ORCC) are present in human hematopoietic myeloblasts. The function of ORCC may be involved in hormone-regulated cell growth, cell volume regulation and immune responses.

Both CFTR and outwardly rectifying chloride channels contribute to cAMP-stimulated whole cell chloride currents.

From whole cell patch-clamp recordings at 35 degrees C utilizing either nystatin perforation or conventional methods with 5 mM MgATP in the pipette solution, it was demonstrated that both cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels and outwardly rectifying Cl- channels (ORCC) contribute to adenosine 3',5'-cyclic monophosphate (cAMP)-activated whole cell Cl- currents in cultured human airway epithelial cells. These results were similar whether recordings were performed on two normal human cell lines or on two cystic fibrosis (CF) cell lines stably complemented with wild-type CF gene. These results were obtained by exploiting dissimilar biophysical properties of CFTR and ORCC currents such as the degree of rectification of the current-voltage relationship, the difference in sensitivity to Cl- channel-blocking drugs such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), calixarenes, and diphenylamine carboxylic acid (DPC), and the opposing Cl- relative to I- permeabilities of the two channels. In normal cells or complemented CF cells, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate stimulated outwardly rectifying whole cell Cl- currents. Addition of DIDS in the presence of cAMP inhibited the outwardly rectifying portion of the cAMP-activated Cl- current. The remaining cAMP-activated, DIDS-insensitive, linear CFTR Cl- current was inhibited completely by DPC. Additional results showed that not only do ORCC and CFTR Cl- channels contribute to cAMP-activated Cl- currents in airway epithelial cells where wild-type CFTR is expressed but that both channels fail to respond to cAMP in delta F508-CFTR-containing CF airway cells. We conclude that CFTR not only functions as a cAMP-regulated Cl- channel in airway epithelial cells but also controls the regulation of ORCC.

Outwardly rectifying chloride channels and CF: a divorce and remarriage.

Outwardly rectifying Cl- channels were originally thought to be the central element in cystic fibrosis. The role of these channels in CF was questioned to such an extent that doubts were raised about the validity of the original experiments. Recent data reestablishes a role for outwardly rectifying Cl- channels (ORCC) in CF and suggests that the protein encoded by the CF gene, the cystic fibrosis transmembrane regulator (CFTR), can effect the regulation of more than one channel in the airway. This minireview deals with the rise, fall, and resurrection of the role of outwardly rectifying Cl- channels in CF.