ClC-5 chloride channel and kidney stones: what is the link?

Nephrolithiasis is one of the most common diseases in the Western world. The disease manifests itself with intensive pain, sporadic infections, and, sometimes, renal failure. The symptoms are due to the appearance of urinary stones (calculi) which are formed mainly by calcium salts. These calcium salts precipitate in the renal papillae and/or within the collecting ducts. Inherited forms of nephrolithiasis related to chromosome X (X-linked hypercalciuric nephrolithiasis or XLN) have been recently described. Hypercalciuria, nephrocalcinosis, and male predominance are the major characteristics of these diseases. The gene responsible for the XLN forms of kidney stones was cloned and characterized as a chloride channel called ClC-5. The ClC-5 chloride channel belongs to a superfamily of voltage-gated chloride channels, whose physiological roles are not completely understood. The objective of the present review is to identify recent advances in the molecular pathology of nephrolithiasis, with emphasis on XLN. We also try to establish a link between a chloride channel like ClC-5, hypercalciuria, failure in urine acidification and protein endocytosis, which could explain the symptoms exhibited by XLN patients.

Physiological implications of the regulation of vacuolar H+-ATPase by chloride ions

Vacuolar H+-ATPase is a large multi-subunit protein that mediates ATP-driven vectorial H+ transport across the membranes. It is widely distributed and present in virtually all eukaryotic cells in intracellular membranes or in the plasma membrane of specialized cells. In subcellular organelles, ATPase is responsible for the acidification of the vesicular interior, which requires an intraorganellar acidic pH to maintain optimal enzyme activity. Control of vacuolar H+-ATPase depends on the potential difference across the membrane in which the proton ATPase is inserted. Since the transport performed by H+-ATPase is electrogenic, translocation of H+-ions across the membranes by the pump creates a lumen-positive voltage in the absence of a neutralizing current, generating an electrochemical potential gradient that limits the activity of H+-ATPase. In many intracellular organelles and cell plasma membranes, this potential difference established by the ATPase gradient is normally dissipated by a parallel and passive Cl- movement, which provides an electric shunt compensating for the positive charge transferred by the pump. The underlying mechanisms for the differences in the requirement for chloride by different tissues have not yet been adequately identified, and there is still some controversy as to the molecular identity of the associated Cl--conducting proteins. Several candidates have been identified: the ClC family members, which may or may not mediate nCl-/H+ exchange, and the cystic fibrosis transmembrane conductance regulator. In this review, we discuss some tissues where the association between H+-ATPase and chloride channels has been demonstrated and plays a relevant physiologic role.

Transmembrane topology of a CLC chloride channel

CLC chloride channels form a large and conserved gene family unrelated to other channel proteins. Knowledge of the transmembrane topology of these channels is important for understanding the effects of mutations found in human myotonia and inherited hypercalciuric kidney stone diseases and for the interpretation of structure–function studies. We now systematically study the topology of human ClC-1, a prototype CLC channel that is defective in human myotonia. Using a combination of in vitro glycosylation scanning and protease protection assays, we show that both N and C termini face the cytoplasm and demonstrate the presence of 10 (or less likely 12) transmembrane spans. Difficult regions were additionally tested by inserting cysteines and probing the effect of cysteine-modifying reagents on ClC-1 currents. The results show that D3 crosses the membrane and D4 does not, and that L549 between D11 and D12 is accessible from the outside. Further, since the modification of cysteines introduced between D11 and D12 and at the extracellular end of D3 strongly affect ClC-1 currents, these regions are suggested to be important for ion permeation.

Cystic fibrosis gene encodes a cAMP-dependent chloride channel in heart.

cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significantly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression of CFTR protein and an endogenous chloride channel in native cardiac myocytes.

Comparative surface accessibility of a pore-lining threonine residue (T6') in the glycine and GABA(A) receptors

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6') common to both the glycine receptor (GlyR) and gamma-aminobutyric acid, type A receptor (GABAAR) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter, and the main pore blocker binding site. Despite their high amino acid sequence homologies and common role in conducting chloride ions, recent studies have suggested that the GlyRs and GABA(A)Rs have divergent open state pore structures at the 6' position. When both the human alpha1(T6'C) homomeric GlyR and the rat alpha1(T6'C)beta1(T6'C) heteromeric GABA(A)R were expressed in human embryonic kidney 293 cells, their 6' residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidizing agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABA(A)R pore structure at the 6' level may vary between different expression systems.

Comparative surface accessibility of a pore-lining threonine residue (T6') in the glycine and GABA(A) receptors

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6’) common to both the glycine receptor (GlyR) and GABAA receptor (GABAAR) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter and the main pore blocker binding site. Recent studies have suggested that the GlyRs and GABAARs have divergent open state pore structures at the 6’ position. When both the human a1T6’C homomeric GlyR and the rat a1T6’Cb1T6’C heteromeric GABAAR were expressed in HEK293 cells, their 6’ residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidising agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABAAR pore structure at the 6’ level may vary between different expression systems.

No evidence for inhibition of ENaC through CFTR-mediated release of ATP

Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl- secretion and inhibit amiloride-sensitive Na+ transport. CFTR has been suggested to conduct adenosine 5'-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na+ channels (ENaC) could be by release of ATP or uridine 5'-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y(2) receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl- channel blocker 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl- transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N-2,2'-O-dibutyrylguanosine 3',5-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl-. (C) 2002 Elsevier Science B.V. All rights reserved.

Asymmetric contribution of α and β subunits to the activation of αβ heteromeric glycine receptors

This study investigated the role of beta subunits in the activation of alphabeta heteromeric glycine receptor (GlyR) chloride channels recombinantly expressed in HEK293 cells. The approach involved incorporating mutations into corresponding positions in alpha and beta subunits and comparing their effects on receptor function. Although cysteine-substitution mutations to residues in the N-terminal half of the alpha subunit M2-M3 loop dramatically impaired the gating efficacy, the same mutations exerted little effect when incorporated into corresponding positions of the beta subunit. Furthermore, although the alpha subunit M2-M3 loop cysteines were modified by a cysteine-specific reagent, the corresponding beta subunit cysteines showed no evidence of reactivity. These observations suggest structural or functional differences between alpha and beta subunit M2-M3 loops. In addition, a threonine-->leucine mutation at the 9' position in the beta subunit M2 pore-lining domain dramatically increased the glycine sensitivity. By analogy with the effects of the same mutation in other ligand-gated ion channels, it was concluded that the mutation affected the GlyR activation mechanism. This supports the idea that the GlyR beta subunit is involved in receptor gating. In conclusion, this study demonstrates that beta subunits contribute to the activation of the GlyR, but that their involvement in this process is significantly different to that of the alpha subunit.

Benzodiazepine receptor ligand influences on learning: an endogenous modulatory mechanism mediated by benzodiazepines possibly of alimentary origin

In rats pre-but not post-training ip administration of either flumazenil, a central benzodiazepine (BSD) receptor antagonist, or of n-butyl-B-carboline-carboxylate (BCCB), an inverse agonist, enhanced retention of inhibitory avoidance learning. Flumazenil vlocked the enhancing effect of BCCB, and the inhibitory effect of the BZD agonists clonazepam and diazepam also given pre-training. Post-training administration of these drugs had no effects. The peripheral BZD receptor agonist/chloride channel blocker Ro5-4864 had no effect on the inhibitory avoidance task when given ip prior to training, buth it caused enhancement when given immediately post-training either ip or icv. This effect was blocked by PK11195, a competitive antagonist of Ro5-4864. These results suggest that ther is an endogenous mechanism mediated by BZD agonists, which is sensitive to inverse agonists and that normally down-regulates the formation of memories through a mechanism involving GABA-A receptors and the corresponding chloride channels. The most likely agonists for the endogenous mechanism suggested are the diazepam-like BZDs found in brain whose origin is possibly alimentary. Levels of these BZDs in the cortex were found to sharply decrease after inhibitory acoidance training or mere exposure to the training apparatus.

Vasopressin-stimulated CFTR Cl- currents are increased in the renal collecting duct cells of a mouse model of Liddle's syndrome.

Liddle's syndrome is a genetic form of hypertension linked to Na(+) retention caused by activating mutations in the COOH terminus of the beta or gamma subunit of the epithelial sodium channel (ENaC). In this study, we used the short-circuit current (I(sc)) method to investigate the effects of deamino-8-d-arginine vasopressin (dDAVP) on Na(+) and Cl(-) fluxes in primary cultures of cortical collecting ducts (CCDs) microdissected from the kidneys of mice with Liddle's syndrome carrying a stop codon mutation, corresponding to the beta-ENaC R(566) stop mutation (L) found in the original pedigree. Compared to wild-type (+/+) CCD cells, untreated L/+ and L/L CCD cells exhibited 2.7- and 4.2-fold increases, respectively, in amiloride-sensitive (Ams) I(sc), reflecting ENaC-dependent Na(+) absorption. Short-term incubation with dDAVP caused a rapid and significant increase (approximately 2-fold) in Ams I(sc) in +/+, but not in L/+ or L/L CCD cells. In sharp contrast, dDAVP induced a greater increase in 5-nitro-2-(3-phenylpropamino)benzoate (NPPB)-inhibited apical Cl(-) currents in amiloride-treated L/L and L/+ cells than in their +/+ counterparts. I(sc) recordings performed under apical ion substituted conditions revealed that the dDAVP-stimulated apical secretion of Cl(-), which was absent in cultured CCDs lacking CFTR, was 1.8-fold greater in L/+ and 3.7-fold greater in L/L CCD cells than in their +/+ CCD counterparts. After the basal membrane had been permeabilized with nystatin and a basal-to-apical Cl(-) gradient had been imposed, dDAVP also stimulated larger Cl(-) currents across L/L and L/+ CCD layers than +/+ CCD layers. These findings demonstrate that vasopressin stimulates greater apical CFTR Cl(-) conductance in the renal CCD cells of mice with Liddle's syndrome than in wild-type mice. This effect could contribute to the enhanced NaCl reabsorption observed in the distal nephron of patients with Liddle's syndrome.

Allelic variation in the VMD2 gene in best disease and age-related macular degeneration.

PURPOSE: To assess the allelic variation of the VMD2 gene in patients with Best disease and age-related macular degeneration (AMD). METHODS: Three hundred twenty-one AMD patients, 192 ethnically similar control subjects, 39 unrelated probands with familial Best disease, and 57 unrelated probands with the ophthalmoscopic findings of Best disease but no family history were screened for sequence variations in the VMD2 gene by single-strand conformation polymorphism (SSCP) analysis. Amplimers showing a bandshift were reamplified and sequenced bidirectionally. In addition, the coding regions of the VMD2 gene were completely sequenced in six probands with familial Best disease who showed no SSCP shift. RESULTS: Forty different probable or possible disease-causing mutations were found in one or more Best disease or AMD patients. Twenty-nine of these variations are novel. Of the 39 probands with familial Best disease, mutations were detected in all 39 (33 by SSCP and 6 by DNA sequencing). SSCP screening of the 57 probands with a clinical diagnosis of Best disease but no family history revealed 16 with mutations. Mutations were found in 5 of 321 AMD patients (1.5%), a fraction that was not significantly greater than in control individuals (0/192, 0%). CONCLUSIONS: Patients with the clinical diagnosis of Best disease are significantly more likely to have a mutation in the VMD2 gene if they also have a positive family history. These findings suggest that a small fraction of patients with the clinical diagnosis of AMD may actually have a late-onset variant of Best disease, whereas at the same time, a considerable fraction of isolated patients with the ophthalmoscopic features of Best disease are probably affected with some other macular disease.

Cells derived from the coelomic epithelium contribute to multiple gastrointestinal tissues in mouse embryos.

Gut mesodermal tissues originate from the splanchnopleural mesenchyme. However, the embryonic gastrointestinal coelomic epithelium gives rise to mesenchymal cells, whose significance and fate are little known. Our aim was to investigate the contribution of coelomic epithelium-derived cells to the intestinal development. We have used the transgenic mouse model mWt1/IRES/GFP-Cre (Wt1(cre)) crossed with the Rosa26R-EYFP reporter mouse. In the gastrointestinal duct Wt1, the Wilms' tumor suppressor gene, is specific and dynamically expressed in the coelomic epithelium. In the embryos obtained from the crossbreeding, the Wt1-expressing cell lineage produces the yellow fluorescent protein (YFP) allowing for colocalization with differentiation markers through confocal microscopy and flow cytometry. Wt1(cre-YFP) cells were very abundant throughout the intestine during midgestation, declining in neonates. Wt1(cre-YFP) cells were also transiently observed within the mucosa, being apparently released into the intestinal lumen. YFP was detected in cells contributing to intestinal vascularization (endothelium, pericytes and smooth muscle), visceral musculature (circular, longitudinal and submucosal) as well as in Cajal and Cajal-like interstitial cells. Wt1(cre-YFP) mesenchymal cells expressed FGF9, a critical growth factor for intestinal development, as well as PDGFRα, mainly within developing villi. Thus, a cell population derived from the coelomic epithelium incorporates to the gut mesenchyme and contribute to a variety of intestinal tissues, probably playing also a signaling role. Our results support the origin of interstitial cells of Cajal and visceral circular muscle from a common progenitor expressing anoctamin-1 and SMCα-actin. Coelomic-derived cells contribute to the differentiation of at least a part of the interstitial cells of Cajal.

Is GABA involved in regulating plant growth and development? /

Rapid and large accumulation of GABA (y-aminobutyric acid) in response to a number of plant stresses has been well documented. But the role(s) of GABA in plants is not well defined. In recent years, the possibility of GABA involvement in regulating plant growth and development has been raised. In the present study, this possibility was examined. First, to rapidly and accurately determine GABA levels in plant tissues, a spectrometric method for GABA determination was developed based on a commercially available enzyme Gabase. Seventy mM LaCb almost completely removed water-soluble pigments from plant tissues which greatly interfere with the absorbance reading at 340nm. Inactivation of GAD (glutamate decarboxylase) by immediately adding methanol to a frozen plant tissue powder was suggested to prevent GABA production during extraction. The recovery of GABA with this method was approximately 100%. Second, the relationship between GABA levels and hypocotyl elongation in soybean seedlings was analyzed using different approaches to regulate in vivo GABA levels and the elongation of hypocotyls. The following major observations were made. (1) Mechanical stimulation by stroking elevated GABA levels and concurrently induced a rapid and significant reduction in hypocotyl elongation. (2) External GABA was demonstrated to penetrate into the hypocotyls using '*C-GABA. Application of external GABA elevated in vivo GABA levels, but failed to inhibit hypocotyl elongation. (3) LaCla and blue light irradiation caused an inhibition in the elongation of dark-grown hypocotyls, whereas GABA levels were not significantly affected. (4) Ca^was suggested to be involved in the signal transduction pathway leading from mechanical stimulation to GABA production, as indicated by the ability of La'* to inhibit GABA production in stimulated hypocotyls. (5) Bicuculline, saclofen and baclofen (agonists and antagonists of GABA receptors in animals) had no effect on hypocotyl elongation. It might indicate that GABA-binding components which are structurally similar to animal GABA receptors and functionally capable of regulating plant growth may not exist in plants. Therefore, the conclusion was drawn that GABA alone is not sufficient to inhibit hypocotyl elongation. Third, chloride influx in isolated Asparagus cells was enhanced by lOmM GABA during a 3 hour incubation, but the effect was not specific for GABA. Chloride efflux was not influenced by GABA. Both influx and efflux of chloride were significantly inhibited by NPPB, a chloride channel blocker. These results suggest that GABA does not influence the activity of plant chloride channels.

Transport d’iode par le transporteur de sodium/acide monocarboxylique SMCT1

Le transporteur de Na+/ acide monocarboxylique sensible à l’ibuprofène (SMCT1) est exprimé dans la membrane apicale de plusieurs épithélia. Son rôle physiologique dans la glande thyroïde reste cependant obscur mais on présume qu’il pourrait agir comme un transporteur apical d’iode nécessaire pour la synthèse des hormones thyroïdiennes. Récemment, on a montré que SMCT1 possède un courant de fuite anionique sensible à [Na+]e qui permettrait de transporter l’iode de façon électrogénique. Cependant, un efflux d’iode sensible à l’ibuprofène, mais indépendant de la [Na+]e a été aussi observé sur des cultures primaires des thyrocytes porcins, suggérant un autre mécanisme de transport d’iode par SMCT1. Ce travail vise à comprendre les caractéristiques de ce genre de transport en utilisant comme modèle d’expression les ovocytes de Xenopus laevis. Les résultats obtenus des essais de captation d’iode radioactif montrent que SMCT1 présente un transport d’iode sensible à l’ibuprofène de l’ordre de 30nmol/ovocyte/h. Si ce transport est non saturable en iode (0-100 mM), il nécessite du Na+ dans la solution externe. En effet, le remplacement du Na+ extracellulaire par le NMDG inhibe complètement le transport. En outre, on s’est intéressé à exclure la possibilité de différents artefacts. En ayant trouvé que la grande majorité de l’iode radioactif se trouve dans la partie soluble de l’ovocyte, on exclut une liaison non spécifique de l’iode à la membrane cellulaire. Cependant, une bonne proportion de l’iode transporté pourrait être liée à des protéines à l’intérieur de l`ovocyte. En effet, on observe une réduction du transport d’iode dans les ovocytes exprimant SMCT1 de 81,6 ± 2 % en présence de 2 % BSA dans la solution extracellulaire. Également, on écarte la possibilité que le transport d’iode soit le résultat de la surexpression de protéines de transport endogènes dont les canaux chlore. Le transport d’iode semble spécifique à l’expression de SMCT1 et de manière intéressante à l’expression d’un autre transporteur de monocarboxylates, MCT1. L’analyse de l’ensemble des essais, y compris le fait que l’amplitude du transport observé est 20 fois plus grande que celle du courant de fuite nous mène à proposer que SMCT1 puisse transporter l’iode de façon électroneutre. Cependant, le mécanisme par lequel ceci est accompli n’est pas évident à identifier. L’utilisation d’un autre modèle cellulaire serait surement utile pour répondre à cette question.