X-ray crystal structures have been previously determined for three CLC-type transporter homologues, but the absolute unitary transport rate is known for only one of these. The Escherichia coli Cl(-)/H(+) antiporter (EC) moves ∼2000 Cl(-) ions/s, an exceptionally high rate among membrane-transport proteins. It is not known whether such rapid turnover is characteristic of ClCs in general or if the E. coli homologue represents a functional outlier. Here, we characterize a CLC Cl(-)/H(+) antiporter from the cyanobacterium Synechocystis sp. PCC6803 (SY) and determine its crystal structure at 3.2 Å resolution. The structure of SY is nearly identical to that of EC, with all residues involved in Cl(-) binding and proton coupling structurally similar to their equivalents in EC. SY actively pumps protons into liposomes against a gradient and moves Cl(-) at ∼20 s(-1), 1% of the EC rate. Electrostatic calculations, used to identify residues contributing to ion binding energetics in SY and EC, highlight two residues flanking the external binding site that are destabilizing for Cl(-) binding in SY and stabilizing in EC. Mutation of these two residues in SY to their counterparts in EC accelerates transport to ∼150 s(-1), allowing measurement of Cl(-)/H(+) stoichiometry of 2/1. SY thus shares a similar structure and a common transport mechanism to EC, but it is by comparison slow, a result that refutes the idea that the transport mechanism of CLCs leads to intrinsically high rates.

The Cl- channel from the Torpedo electric organ, CLC-0, is the prototype of a large gene family of Cl- channels. At the single-channel level, CLC-0 shows a "double-barreled" behavior. Recently it was shown that CLC-0 is a dimer, and it was suggested that each subunit forms a single pore. The two protopores are gated individually by a fast voltage and anion-dependent gating mechanism. A slower common gating mechanism operates on both pores simultaneously. Previously, wild-type/mutant heteromeric channels had been constructed that display a large wild-type pore and small mutant pore. Here we use patch-clamp recording of single wild-type and mutant CLC-0 channels to investigate in detail the dependence of the gating of one protopore on the physically attached neighboring pore. No difference in rate constants of opening and closing of protopores could be found comparing homomeric wild-type and heteromeric wild-type/mutant channels. In addition, detailed kinetic analysis reveals that gating of single subunits is not correlated with the gating of the neighboring subunit. The results are consistent with the view that permeation and fast gating of individual pores are fully independent of the neighboring pore. Because the two subunits are associated in a common protein complex, opening and closing transitions of individual pores are probably due to only small conformational changes in each pore. In addition to the fast and slow gating mechanisms known previously for CLC-0, in the course of this study we occasionally observed an additional gating process that led to relatively long closures of single pores.

The two human CLC Cl(-) channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel beta subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca(2+) and H(+) on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca(2+)](ext) without full saturation up to 50 mM. However, in the absence of Ca(2+), ClC-Ka currents were still 20% of currents in 10 mM [Ca(2+)](ext), demonstrating that Ca(2+) is not strictly essential for opening. Vice versa, ClC-Ka and ClC-Kb were blocked by increasing [H(+)](ext) with a practically complete block at pH 6. Ca(2+) and H(+) act as gating modifiers without changing the single-channel conductance. Dose-response analysis suggested that two protons are necessary to induce block with an apparent pK of approximately 7.1. A simple four-state allosteric model described the modulation by Ca(2+) assuming a 13-fold higher Ca(2+) affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca(2+) and H(+). A mutagenic screen of a large number of extracellularly accessible amino acids identified a pair of acidic residues (E261 and D278 on the loop connecting helices I and J), which are close to each other but positioned on different subunits of the channel, as a likely candidate for forming an intersubunit Ca(2+)-binding site. Single mutants E261Q and D278N greatly diminished and the double mutant E261Q/D278N completely abolished modulation by Ca(2+). Several mutations of a histidine residue (H497) that is homologous to a histidine that is responsible for H(+) block in ClC-2 did not yield functional channels. However, the triple mutant E261Q/D278N/H497M completely eliminated H(+) -induced current block. We have thus identified a protein region that is involved in binding these physiologically important ligands and that is likely undergoing conformational changes underlying the complex gating of CLC-K channels.

Dendritic cells (DC) are characterized by their unique potential to prime naive T cells. Epidermal Langerhans cells (LC), the DC resident in the epidermis, gain this immunostimulatory capacity following Ag contact in vivo or during in vitro culture of epidermal cell suspensions. To analyze differential gene expression in maturing LC, we constructed a highly representative cDNA library of cultivated LC (cLC) in lambda ZAP II containing 18 x 10(6) independent clones. This library was screened with freshly isolated Langerhans cell (fLC)- and cLC-derived probes for cLC-specific cDNAs. The cDNAs identified were sequenced and analyzed by database searches. Two cDNA fragments were identified as fragments of fascin, indicating that fascin is differentially expressed in LC. By competitive RT-PCR, we confirmed that fascin is highly expressed in cLC cultivated for 1, 2, and 3 days, while no signals were obtained with fLC. Western blot and immunofluorescence analysis revealed cLC-specific expression of fascin on the protein level as well. Fascin is known to be involved in the organization of the actin cytoskeleton in cytoplasmatic extensions of nerve growth cones. Its differential expression in maturing LC coincides with the formation of numerous dendritic projections in LC. Their formation was inhibited by incubation of LC with fascin antisense oligonucleotides during cultivation. Therefore, we conclude that fascin is necessary for the formation of the dendritic processes of maturing Langerhans cells and may thus influence T cell-LC interaction.

Receptor-mediated endocytosis is a constitutive high capacity pathway for the reabsorption of proteins from the glomerular filtrate by the renal proximal tubule. ClC-5 is a voltage-gated chloride channel found in the proximal tubule where it has been shown to be essential for protein uptake, based on evidence from patients with Dent's disease and studies in ClC-5 knockout mice. To further delineate the role of ClC-5 in albumin uptake, we performed a yeast two-hybrid screen with the C-terminal tail of ClC-5 to identify any interactions of the channel with proteins involved in endocytosis. We found that the C-terminal tail of ClC-5 bound the actin depolymerizing protein, cofilin, a result that was confirmed by GST-fusion pulldown assays. In cultured proximal tubule cells, cofilin was distributed in nuclear, cytoplasmic, and microsomal fractions and co-localized with ClC-5. Phosphorylation of cofilin by overexpressing LIM kinase 1 resulted in a stabilization of the actin cytoskeleton. Phosphorylation of cofilin in two proximal tubule cell models (porcine renal proximal tubule and opossum kidney) was also accompanied by a pronounced inhibition of albumin uptake. This study identifies a novel interaction between the C-terminal tail of ClC-5 and cofilin, an actin-associated protein that is crucial in the regulation of albumin uptake by the proximal tubule.

The proper functioning of the Cl(-) channel, ClC-5, is essential for the uptake of low molecular mass proteins through receptor-mediated endocytosis in the proximal tubule. Dent's disease patients with mutant ClC-5 channels and ClC-5 knockout (KO) mice both have low molecular mass proteinuria. To further understand the function of ClC-5, endocytosis was studied in LLC-PK(1) cells and primary cultures of proximal tubule cells from wild-type (WT) and ClC-5 KO kidneys. Endocytosis in the proximal tubule cells from KO mice was reduced compared with that in WT animals. Endocytosis in WT but not in KO cells was inhibited by bafilomycin A-1 and Cl(-) depletion, whereas endocytosis in both WT and KO cells was inhibited by the NHE3 blocker, S3226. Infection with adenovirus containing WT ClC-5 rescued receptor-mediated endocytosis in KO cells, whereas infection with any of the three disease-causing mutants, myc-W22G-ClC-5, myc-S520P-ClC-5, or myc-R704X-ClC-5, did not. WT and the three mutants all trafficked to the apical surface, as assessed by surface biotinylation. WT-ClC-5 and the W22G mutant were internalized similarly, whereas neither the S520P nor the R704X mutants was. These data indicate that ClC-5 is important for Cl(-) and proton pump-mediated endocytosis. However, not all receptor-mediated endocytosis in the proximal tubule is dependent on ClC-5. There is a significant fraction that can be inhibited by an NHE3 blocker. Our data from the mutants suggest that defective targeting and trafficking of mutant ClC-5 to the endosomes are a major determinant in the lack of normal endocytosis in Dent's disease.

ClC chloride channels (ClCs) can be classified into two groups in terms of their cellular localizations: ClCs present in the plasma membranes and those residing in intracellular organelles. Members of the latter group, including ClC-3, ClC-4, ClC-5, ClC-6, and ClC-7, are often co-expressed in a variety of cell types in many organs. Although the localization of individual channels within cells has been investigated, the degree of overlap between the locations of different ClCs in the same cell has not been clarified. To address this question, different combinations of ClCs, engineered to encode specific epitope tags (FLAG or HA), were either transiently or stably transfected into HEK293 cells, and we then compared the intracellular localization of the expressed channel proteins by immunofluorescence microscopy. Immunofluorescence images of the alternatively labeled channels clearly showed significant co-localization between all pair-wise combinations of ClCs. In particular, ClC-3, ClC-4, and ClC-5 showed a high degree of co-localization. As a significant degree of co-localization between ClCs was observed, we used co-immunoprecipitation to evaluate oligomer formation, and found that each ClC tested could form homo-oligomers, and that any pair-wise combination of ClC-3, ClC-4, and ClC-5 could also form hetero-oligomers. Neither ClC-6 nor ClC-7 was co-precipitated with any other channel protein. These results suggest that within cells ClC-3, ClC-4, and ClC-5 may have combinatorial functions, whereas ClC-6 and ClC-7 are more likely to function as homo-oligomers.

Changes in synaptic activity alter quantal size, but the relative roles of presynaptic and postsynaptic cells in these changes are only beginning to be understood. We examined the mechanism underlying increased quantal size after block of synaptic activity at the mammalian neuromuscular junction in vivo. We found that changes in neither acetylcholinesterase activity nor acetylcholine receptor density could account for the increase. By elimination, it appears likely that the site of increased quantal size after chronic block of activity is presynaptic and involves increased release of acetylcholine. We used mice with muscle hyperexcitability caused by mutation of the ClC-1 muscle chloride channel to examine the role of postsynaptic activity in controlling quantal size. Surprisingly, quantal size was increased in ClC mice before block of synaptic activity. We examined the mechanism underlying increased quantal size in ClC mice and found that it also appeared to be located presynaptically. When presynaptic activity was completely blocked in both control and ClC mice, quantal size was large in both groups despite the higher level of postsynaptic activity in ClC mice. This suggests that postsynaptic activity does not regulate quantal size at the neuromuscular junction. We propose that presynaptic activity modulates quantal size at the neuromuscular junction by modulating the amount of acetylcholine released from vesicles.

Voltage-gated Cl- channels belonging to the ClC family exhibit unique properties of ion permeation and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl- channel (hClC-1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or intracellular I- and related anions. Extracellular and intracellular I- exert blocking actions on hClC-1 currents that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide (Br-) and polyatomic (SCN-, NO3-, CH3SO3-) anions. In addition, I- block is accompanied by gating alterations that differ depending on which side of the membrane the blocker is applied. External I- causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating, nondeactivating), while internal I- slows deactivation. These different effects on gating properties can be used to distinguish two functional ion binding sites within the hClC-1 pore. We determined KD values for I- block in three distinct kinetic states and found that binding of I- to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I- binding suggest that conformational changes affecting the two ion binding sites occur during gating transitions. These results have implications for understanding mechanisms of ion selectivity in hClC-1, and for defining the intimate relationship between gating and permeation in ClC channels.

Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

Endosome trafficking and function require acidification by the vacuolar ATPase (V-ATPase). Electrogenic proton (H+) transport reduces the pH and creates a net positive charge in the endosomal lumen. Concomitant chloride (Cl-) influx has been proposed to occur via ClC Cl-=H+ exchangers. This maintains charge balance and drives Cl- accumulation, which may itself be critical to endosome function. Production of reactive oxygen species (ROS) in response to cytokines occurs within specialized endosomes that form in response to receptor occupation. ROS production requires an NADPH oxidase (Nox) and the ClC-3 Cl-=H+ exchanger. Like the V-ATPase, Nox activity is highly electrogenic, but separates charge with an opposite polarity (lumen negative). Here we review established paradigms of early endosomal ion transport focusing on the relation between the V-ATPase and ClC proteins. Electrophysiologic constraints on Nox-mediated vesicular ROS production are then considered. The potential for ClC-3 to participate in charge neutralization of both proton (V-ATPase) and electron (Nox) transport is discussed. It is proposed that uncompensated charge separation generated by Nox enzymatic activity could be used to drive secondary transport into negatively charged vesicles. Further experimentation will be necessary to establish firmly the biochemistry and functional implications of endosomal ROS production.

Cellular ion homeostasis involves communication between the cytosol and the luminal compartment of organelles. This is particularly critical for metal ions because of their toxic potential. We have identified the yeast homologue of the prokaryotic ArsA protein, the homodimeric ATPase Arr4p, as a protein that binds to the yeast intracellular CLC chloride-transport protein, Gef1p. We show that binding of Arr4p to the C terminus of Gef1p requires the presence of yeast cytosol and is sensitive to a highly specific copper chelator in vitro and in vivo. Copper alone can substitute for cytosol to support the interaction of Arr4p with the C terminus of Gef1p. The migration behavior of Arr4p in nonreducing gel electrophoresis correlates with cellular copper deficiency, repletion, or stress. Our homology model of Arr4p shows that the antimony (arsenic) metal binding site of ArsA is not conserved in Arr4p. The model suggests that a pair of cysteines, Cys285 and Cys288, is located in the interface of the Arr4p dimer. These residues are required for Arr4p homodimerization and for binding to the C terminus of Gef1p. Whereas both proteins are required for normal growth under iron-limiting conditions, they play opposite roles when copper and heat stress are combined in an alkaline environment. Under these conditions, deltagef1 cells grow much better than wild type yeast, whereas deltaarr4 cells are unable to grow. Comparison of the deltaarr4 with the deltaarr4deltagef1 strain suggests that Arr4p antagonizes the function of Gef1p.

Dissolution of the inorganic phase of bone by the osteoclasts mediated by V-ATPase and ClC-7 is a prerequisite for bone resorption. Inhibitors of osteoclastic V-ATPase or ClC-7 are novel approaches for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays, diphyllin was identified. We characterized diphyllin with respect to the pharmacological effects on osteoclasts.

BACKGROUND

Osteoclastic acidification of the resorption lacuna and bone resorption requires activity of both V-ATPase and the chloride channel ClC-7. Inhibition of these processes represents a novel approach for treatment of bone metabolic disorders. We identified diphyllin, a novel inhibitor of V-ATPase, and characterized this natural compound with respect to activity in human osteoclasts.

METHODS

Diphyllin was tested in the acid influx assay and V-ATPase assay using bovine chromaffin granules. Human osteoclasts were generated from CD14+ monocytes cultured with macrophage-colony stimulating factor (M-CSF) and RANKL. The effect of diphyllin on lysosomal acidification in human osteoclasts was studied using acridine orange. The effect of diphyllin on bone resorption by osteoclasts was measured as release of C-terminal cross-linked telopeptide of type I collagen (CTX-I) and calcium into the supernatants and by scoring pit area. Osteoclast number, TRACP activity, and cell viability were measured. Furthermore, the effect of diphyllin on bone nodule formation was tested using the mouse osteoblast cell line MC3T3-E1.

RESULTS

In the acid influx assay, diphyllin potently inhibited the acid influx (IC50 = 0.6 nM). We found that diphyllin inhibited V-ATPase with an IC50 value of 17 nM, compared with 4 nM for bafilomycin A1. Moreover, diphyllin dose-dependently inhibited lysosomal acidification in human osteoclasts. Furthermore, we found that diphyllin inhibited human osteoclastic bone resorption measured by CTX-I (IC50 = 14 nM), calcium release, and pit area, despite increasing TRACP activity, numbers of osteoclasts, and cell viability. Finally, diphyllin showed no effect on bone formation in vitro, whereas bafilomycin A1 was toxic.

CONCLUSIONS

We identified a natural compound that potently inhibits V-ATPase and thereby lysosomal acidification in osteoclasts, which leads to abrogation of bone resorption. Because recent studies indicate that inhibition of the osteoclastic acidification leads to inhibition of resorption without inhibiting formation, we speculate that diphyllin is a potential novel treatment for bone disorders involving excessive resorption.

We have cloned ClC-3B, a novel alternative splicing variant of ClC-3 (ClC-3A) that is expressed predominantly in epithelial cells. ClC-3B has a different, slightly longer C-terminal end than ClC-3A and contains a consensus motif for binding to the second PDZ (PSD95/Dlg/ZO-1) domain of the epithelium-specific scaffolding protein EBP50. Both in vitro and in vivo binding assays demonstrate interaction between ClC-3B and EBP50. C127 mouse mammary epithelial cells transfected with ClC-3B alone showed diffuse immunoreactivity for ClC-3B in the cytoplasmic region. In contrast, when EBP50 was cotransfected with ClC-3B, strong immunoreactivity for ClC-3B appeared at the leading edges of membrane ruffles. Patch-clamp experiments revealed that cotransfection of ClC-3B and EBP50 resulted in a remarkable increase in outwardly rectifying Cl- channel (ORCC) activities at the leading edges of membrane ruffles in C127 cells. The electrophysiological properties of the ClC-3B-induced ORCCs are similar to those of ORCCs described in native epithelial cells. When cystic fibrosis transmembrane conductance regulator (CFTR) was cotransfected with ClC-3B and EBP50, ClC-3B-dependent ORCCs were activated via the protein kinase A-dependent pathway. These findings indicate that ClC-3B is itself a CFTR-regulated ORCC molecule or its activator.

ClC-3, a member of the large superfamily of ClC voltage-dependent Cl(-) channels, has been proposed as a molecular candidate responsible for volume-sensitive osmolyte and anion channels (VSOACs) in some cells, including heart and vascular smooth muscle. However, the reported presence of native VSOACs in at least two cell types from transgenic ClC-3 disrupted (Clcn3(-/-)) mice casts considerable doubt on this proposed role for ClC-3. We compared several properties of native VSOACs and examined mRNA transcripts and membrane protein expression profiles in cardiac and pulmonary arterial smooth muscle cells from Clcn3(+/+) and Clcn3(-/-) mice to: (1) test the hypothesis that native VSOACs are unaltered in cells from Clcn3(-/-) mice, and (2) test the possibility that targeted inactivation of the Clcn3 gene using a conventional murine global knock-out approach may result in compensatory changes in expression of other membrane proteins. Our experiments demonstrate that VSOAC currents in myocytes from Clcn3(+/+) and Clcn3(-/-) mice are remarkably similar in terms of activation and inactivation kinetics, steady-state current densities, rectification, anion selectivity (I(-)>> Cl(->>) Asp(-)) and sensitivity to block by glibenclamide, niflumic acid, DIDS and extracellular ATP. However, additional experiments revealed several significant differences in other fundamental properties of native VSOACs recorded from atrial and smooth muscle cells from Clcn3(-/-) mice, including: differences in regulation by endogenous protein kinase C, differential sensitivity to block by anti-ClC-3 antibodies, and differential sensitivities to [ATP](i) and free [Mg(2+)](i). These results suggest that in response to Clcn3 gene deletion, there may be compensatory changes in expression of other proteins that alter VSOAC channel subunit composition or associated regulatory subunits that give rise to VSOACs with different properties. Consistent with this hypothesis, in atria from Clcn3(-/-) mice compared to Clcn3(+/+) mice, quantitative analysis of ClC mRNA expression levels revealed significant increases in transcripts for ClC-1, ClC-2, and ClC-3, and protein expression profiles obtained using two-dimensional polyacrylamide gel electrophoresis revealed complex changes in at least 35 different unidentified membrane proteins in cells from Clcn3(-/-) mice. These findings emphasize that caution needs to be exercised in simple attempts to interpret the phenotypic consequences of conventional global Clcn3 gene inactivation.

In smooth muscle cells (SMCs), the intracellular chloride ion (Cl−) concentration is high due to accumulation by Cl−/HCO3− exchange and Na+–K+–Cl− cotransportation. The equilibrium potential for Cl− (ECl) is more positive than physiological membrane potentials (Em), with Cl− efflux inducing membrane depolarization. Early studies used electrophysiology and nonspecific antagonists to study the physiological relevance of Cl− channels in SMCs. More recent reports have incorporated molecular biological approaches to identify and determine the functional significance of several different Cl− channels. Both "classic" and cGMP-dependent calcium (Ca2+)-activated (ClCa) channels and volume-sensitive Cl− channels are present, with TMEM16A/ANO1, bestrophins, and ClC-3, respectively, proposed as molecular candidates for these channels. The cystic fibrosis transmembrane conductance regulator (CFTR) has also been described in SMCs. This review will focus on discussing recent progress made in identifying each of these Cl− channels in SMCs, their physiological functions, and contribution to diseases that modify contraction, apoptosis, and cell proliferation.

OBJECTIVE

To assess the percentage volumes of filling materials and voids in oval-shaped canals filled with either cold lateral compaction or warm compaction techniques, using micro-computed tomography (micro-CT).

METHODS

Twenty-four single-rooted maxillary premolar teeth with oval-shaped canals were selected and the root canals prepared and assigned to two groups (n = 12), according to the filling technique: cold lateral compaction (CLC) or warm vertical compaction (WVC). Each specimen was scanned using a micro-CT device at an isotropic resolution of 12.5 μm. Percentage volumes of root filling materials and voids were calculated, and data were statistically analysed using Student's t-test and Friedman's test, with a significance level of 5%.

RESULTS

Overall, mean percentage volumes of gutta-percha, sealer and voids were 82.33 ± 3.14, 13.42 ± 2.91 and 4.26 ± 0.74 in the CLC group and 91.73 ± 4.48, 7.70 ± 4.44 and 0.57 ± 0.44 in the WVC group, respectively, with a statistically significant difference between groups (P < 0.05). At the apical level, differences in the percentage volumes of filling materials and voids between groups were not significant (P>> 0.05).

CONCLUSIONS

No root fillings were void free. Warm vertical compaction produced a significantly greater volume of gutta-percha and a significantly lower percentage of voids than those achieved with cold lateral compaction. Distribution of sealer and voids within the root canal space after root filling was unpredictable, irrespective of the technique used.

At least seven different chloride/anion currents have now been identified in astrocytes, oligodendrocytes/Schwann cells, and microglia. Only for two of these currents is the corresponding gene known. One of these genes is not encoding for a chloride channel, but for a class of mitochondria-like pores also found in cell membranes. Astrocytes and oligodendrocytes differ in their resting properties: astrocytes accumulate chloride but do not have a significant permeability. Oligodendrocytes have a close to passive distribution and a significant permeability. Under certain circumstances, astrocytes can express a resting chloride conductance. Reactive and neoplastic astrocytes as well as astrocytes with an altered shape exhibit a resting conductance. The function of these channels certainly involves volume regulation. Other possible functions are potassium homeostasis, migration, proliferation (in microglia), and involvement in spreading depression waves. Of greatest interest are two phenomena discovered in situ: The ClC-2 channel is only found in astrocytic endfeet near blood capillaries adjacent to neuronal GABA(A) receptors. In the supraoptic nucleus of the hypothalamus, there is an osmosensitive astrocytic taurine release. This released taurine interacts with glycine receptors in neighboring neurons, causing inhibition. It is assumed that with the future availability of more in situ, rather than in vitro, studies, an increased number of such complex interactions between glial cells, neurons, and blood vessels will be discovered.

Only a small number of new drugs have recently become available for gastrointestinal (GI) disorders. This is partly because we await outcomes of research into functional bowel disorder aetiology (e.g., role of microbiota) and of trials to control stress- related or painful GI symptoms (e.g., via CRF(1) receptors or beta(3) adrenoceptors). Nevertheless, only the ClC-2 channel activator lubiprostone has recently reached the clinic, joining the 5-HT(3) antagonist alosetron and the long-established 5-HT(4) agonist and D(2) antagonist metoclopramide; tegaserod, a non-selective ligand, was withdrawn. Interestingly, each has shortcomings, providing opportunities for molecules with 5-HT(4) or motilin receptor selectivity, and for new biology via guanylate cyclase C or ghrelin receptor activation. For translation into new drugs, the molecule must have appropriate efficacy, selectivity and pharmacodynamic properties. It is argued that the compound must then be evaluated in conditions where changes in motility are known to exist, before considering more difficult symptomatic conditions such as irritable bowel syndrome (IBS) or functional dyspepsia (FD), where relationships with disordered motility are unclear. Thus, it may be better to begin studying a gastric prokinetic in diabetics requiring improved glucose control, rather than in FD. Notably, new 5-HT(4) receptor agonists are being evaluated firstly as treatments of constipation, not IBS. New antidiarrhoeal agents should be developed similarly. Thus, progression of new drugs may require initial studies in smaller patient populations where clinical outcome is better defined. Only then can disease-related ideas be properly tested and drugs brought forward for these disorders (with high clinical need) and then, if successful for IBS and FD.

Cardiotrophin-like cytokine (CLC), also known as novel neurotrophin-1/B cell stimulating factor-3 (NNT-1/BSF-3), is a recently identified member of the interleukin (IL)-6 family of cytokines that share gp130 as a signal-transducing receptor component. In this study, we demonstrate that CLC is expressed in fetal mouse neuroepithelial cells and has a potential to induce their astrocyte differentiation in a synergistic manner with bone-morphogenetic protein (BMP)-2, which is also expressed in the fetal mouse brain. CLC-stimulation led to promoter activation of the gene for an astrocyte marker, glial fibrillary acidic protein (GFAP), which was clearly inhibited by expression of a dominant negative form of a transcription factor, STAT3, or by introduction of a mutation in a single STAT3-binding site in the promoter, suggesting a critical role of STAT3 in the CLC-induced GFAP transcription. These results suggest that CLC plays a role in astrocyte differentiation via STAT3 activation within the developing brain.

ClC-Ka and ClC-Kb Cl(-) channels are pivotal for renal salt reabsorption and water balance. There is growing interest in identifying ligands that allow pharmacological interventions aimed to modulate their activity. Starting from available ligands, we followed a rational chemical strategy, accompanied by computational modeling and electrophysiological techniques, to identify the molecular requisites for binding to a blocking or to an activating binding site on ClC-Ka. The major molecular determinant that distinguishes activators from blockers is the level of planarity of the aromatic portions of the molecules: only molecules with perfectly coplanar aromatic groups display potentiating activity. Combining several molecular features of various CLC-K ligands, we discovered that phenyl-benzofuran carboxylic acid derivatives yield the most potent ClC-Ka inhibitors so far described (affinity <10 microM). The increase in affinity compared with 3-phenyl-2-p-chlorophenoxy-propionic acid (3-phenyl-CPP) stems primarily from the conformational constraint provided by the phenyl-benzofuran ring. Several other key structural elements for high blocking potency were identified through a detailed structure-activity relationship study. Surprisingly, some benzofuran-based drugs inhibit ClC-Kb with a similar affinity of <10 microM, thus representing the first inhibitors for this CLC-K isoform identified so far. Based on our data, we established a pharmacophore model that will be useful for the development of drugs targeting CLC-K channels.

A novel Cl(-) inward rectifier channel (Cl,ir) encoded by ClC-2, a member of the ClC voltage-gated Cl(-) channel gene superfamily, has been recently discovered in cardiac myocytes of several species. However, the physiological role of Cl,ir channels in the heart remains unknown. In this study we tested the hypothesis that Cl,ir channels may play an important role in cardiac pacemaker activity. In isolated guinea-pig sinoatrial node (SAN) cells, Cl,ir current was activated by hyperpolarization and hypotonic cell swelling. RT-PCR and immunohistological analyses confirmed the molecular expression of ClC-2 in guinea-pig SAN cells. Hypotonic stress increased the diastolic depolarization slope and decreased the maximum diastolic potential, action potential amplitude, APD(50), APD(90), and the cycle-length of the SAN cells. These effects were largely reversed by intracellular dialysis of anti-ClC-2 antibody, which significantly inhibited Cl,ir current but not other pacemaker currents, including the hyperpolarization-activated non-selective cationic "funny" current (I(f)), the L-type Ca(2+) currents (I(Ca,L)), the slowly-activating delayed rectifier I(Ks) and the volume-regulated outwardly-rectifying Cl(-) current (I(Cl,vol)). Telemetry electrocardiograph studies in conscious ClC-2 knockout (Clcn2(-/-)) mice revealed a decreased chronotropic response to acute exercise stress when compared to their age-matched Clcn2(+/+) and Clcn2(+/-) littermates. Targeted inactivation of ClC-2 does not alter intrinsic heart rate but prevented the positive chronotropic effect of acute exercise stress through a sympathetic regulation of ClC-2 channels. These results provide compelling evidence that ClC-2-encoded endogenous Cl,ir channels may play an important role in the regulation of cardiac pacemaker activity, which may become more prominent under stressed or pathological conditions.

Mutations of the human CLCN5 gene, which encodes the CLC-5 Cl(-)/H(+) exchanger, lead to Dent's disease. Mutations result in functional defects that range from moderate reductions to complete loss of whole cell currents, although the severity of the functional defect rarely correlates with the severity of the disease. To further elucidate the basis of CLC-5 mutations causing Dent's disease, we examined the functional and cell biological consequences of seven previously reported missense mutants, utilizing electrophysiological and cell biological techniques. This revealed three classes of Dent's disease-causing CLC-5 mutations. Class 1 mutations lead to endoplasmic reticulum retention and degradation of CLC-5. Class 2 mutations appear to have little effect on subcellular distribution of CLC-5 but cause defective function resulting in severe defects in endosomal acidification. Class 3 mutations lead to alterations in the endosomal distribution of CLC-5 but are otherwise able to support endosomal acidification. Molecular modeling demonstrates a structural basis that may underlie the nature of the defect resulting from each mutation with each class occupying discrete regions of the protein quaternary structure. Thus these results demonstrate that the cell biological consequences of CLC-5 mutations are heterogeneous and can be classified into three major groups and that a correlation between the nature of the defect and the location of the mutation in the structure may be drawn. This model may prove to be useful as a tool to aid in the diagnosis and future therapeutic intervention of the disease.

BACKGROUND

Lubiprostone, used clinically (b.i.d.) to treat constipation, has been reported to increase transepithelial Cl(-) transport in T84 cells by activating ClC-2 channels.

OBJECTIVE

To identify the underlying signaling pathway, we explored the effects of short-term and overnight lubiprostone treatment on second messenger signaling and Cl(-) transport.

METHODS

Cl(-) transport was assessed either as I(sc) across T84 monolayers grown on Transwells and mounted in Ussing chambers or by the iodide efflux assay. [cAMP](i) was measured by enzyme immunoassay, and [Ca(2+)](i) by Fluo-3 fluorescence. Quantitation of apical cell surface CFTR protein levels was assessed by Western blotting and biotinylation with the EZ-Link Sulfo-NHS-LC-LC-Biotin. ClC-2 mRNA level was studied by RT-PCR.

RESULTS

Lubiprostone and the cAMP stimulator, forskolin, caused comparable and maximal increases of I(sc) in T84 cells. The I(sc) effects of lubiprostone and forskolin were each suppressed if the tissue had previously been treated with the other agent. These responses were unaltered even if the monolayers were treated with lubiprostone overnight. Lubiprostone-induced increases in iodide efflux were ~80% of those obtained with forskolin. Lubiprostone increased [cAMP](i). H89, bumetanide, or CFTR(inh)-172 greatly attenuated lubiprostone-stimulated Cl(-) secretion, whereas the ClC-2 inhibitor CdCl(2) did not. Compared to controls, FSK-treatment increased membrane-associated CFTR by 1.9 fold, and lubiprostone caused a 2.6-fold increase in apical membrane CFTR as seen by immunoblotting following cell surface biotinylation.

CONCLUSIONS

Lubiprostone activates Cl(-) secretion in T84 cells via cAMP, protein kinase A, and by increasing apical membrane CFTR protein.