HIV-infected dendritic cells (DC) efficiently transmit infection to CD4+ T cells during the process of T cell activation. To further understand interactions between DC and HIV, cytokine regulation of HIV coreceptors on cultured Langerhans cells (cLC, as prototypes of mature DC) was studied. Expression of cell surface CXCR4 on cLC was up-regulated by IL-4 and TGF-beta1 and inhibited by IFN-alpha, IFN-beta, and IFN-gamma, whereas cytokines did not appreciably regulate CCR5. Changes in cell surface CXCR4 expression on cLC correlated with T cell-tropic (X4)-HIV envelope-mediated syncytium formation and X4-HIV infection levels. A relative increase in the ratio of type 2/type 1 cytokine production, which can occur in HIV disease, may up-regulate CXCR4 expression on mature DC and promote infection by X4 viruses. Importantly, these findings suggest that cytokine dysregulation may be linked to the emergence of X4-HIV strains as HIV-infected individuals progress to AIDS.

Dent's disease is an X-linked renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and eventual renal failure. Various types of mutations in the renal chloride channel gene, CLCN5, have been identified in patients with this disease. We studied a Spanish patient with Dent's disease and found, by polymerase chain reaction amplification of the CLCN5 exons, an abnormally large exon 11. Sequencing analysis revealed that this was attributable to the insertion in codon 650 of an Alu element of the "young" Ya5 subfamily. The Alu element was inserted with the same orientation as the CLCN5 gene and arose de novo on the maternal chromosome. Polymorphism analysis indicated that the insertion occurred in the germline of the maternal grandfather. The presence of a long poly(A) tract and evidence for a 16-bp target-site duplication implied that the Alu element was integrated by retrotransposition. This mutation predicts a truncated ClC-5 protein that lacks part of the carboxy-terminus and is likely to result in loss of function of the chloride channel. Insertions of Alu sequences, which are rarely found in coding regions, have occasionally been reported to cause other genetic diseases. However, this is the first report of a retrotransposon insertion in the CLCN5 gene associated with Dent's disease.

Inactivation of the mainly endosomal 2Cl(-)/H(+)-exchanger ClC-5 severely impairs endocytosis in renal proximal tubules and underlies the human kidney stone disorder Dent's disease. In heterologous expression systems, interaction of the E3 ubiquitin ligases WWP2 and Nedd4-2 with a "PY-motif" in the cytoplasmic C terminus of ClC-5 stimulates its internalization from the plasma membrane and may influence receptor-mediated endocytosis. We asked whether this interaction is relevant in vivo and generated mice in which the PY-motif was destroyed by a point mutation. Unlike ClC-5 knock-out mice, these knock-in mice displayed neither low molecular weight proteinuria nor hyperphosphaturia, and both receptor-mediated and fluid-phase endocytosis were normal. The abundances and localizations of the endocytic receptor megalin and of the Na(+)-coupled phosphate transporter NaPi-2a (Npt2) were not changed, either. To explore whether the discrepancy in results from heterologous expression studies might be due to heteromerization of ClC-5 with ClC-3 or ClC-4 in vivo, we studied knock-in mice additionally deleted for those related transporters. Disruption of neither ClC-3 nor ClC-4 led to proteinuria or impaired proximal tubular endocytosis by itself, nor in combination with the PY-mutant of ClC-5. Endocytosis of cells lacking ClC-5 was not impaired further when ClC-3 or ClC-4 was additionally deleted. We conclude that ClC-5 is unique among CLC proteins in being crucial for proximal tubular endocytosis and that PY-motif-dependent ubiquitylation of ClC-5 is dispensable for this role.

The CLC gene family encodes Cl(-) channels or Cl(-)/H(+) exchangers. While our understanding of their structure-function relationship has greatly benefited from the crystal structure of bacterial homologues, human inherited diseases and knock-out mice were crucial in deciphering their physiological roles. Several vesicular CLC Cl(-)/H(+) exchangers are expressed in the proximal tubule (PT). ClC-5 mutations cause Dent's disease which is associated with low molecular weight proteinuria and kidney stones. ClC-5 knock-out mice revealed impaired endocytosis as the primary defect in Dent's disease. It extends to receptor-mediated and fluid-phase endocytosis and entails changes in calciotropic hormones that result in kidney stones. No renal functions could be assigned so far to ClC-3 and ClC-4, which are also expressed in PTs. Loss of ClC-7 or its beta-subunit Ostm1 entails lysosomal storage in the PT, in addition to the neuronal lysosomal storage and osteopetrosis that are the hallmarks of ClC-7/Ostm1 loss in mice and men.

"Mitotic cell rounding" describes the rounding of mammalian cells before dividing into two daughter cells. This shape change requires coordinated cytoskeletal contraction and changes in osmotic pressure. While considerable research has been devoted to understanding mechanisms underlying cytoskeletal contraction, little is known about how osmotic gradients are involved in cell division. Here we describe cytoplasmic condensation preceding cell division, termed "premitotic condensation" (PMC), which involves cells extruding osmotically active Cl(-) via ClC-3, a voltage-gated channel/transporter. This leads to a decrease in cytoplasmic volume during mitotic cell rounding and cell division. Using a combination of time-lapse microscopy and biophysical measurements, we demonstrate that PMC involves the activation of ClC-3 by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in human glioma cells. Knockdown of endogenous ClC-3 protein expression eliminated CaMKII-dependent Cl(-) currents in dividing cells and impeded PMC. Thus, kinase-dependent changes in Cl(-) conductance contribute to an outward osmotic pressure in dividing cells, which facilitates cytoplasmic condensation preceding cell division.

Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype is associated with CF. Recent studies have shown that the level of CFTR mRNA in mouse kidney approaches that found in lung. CFTR is particularly abundant in the apical area of proximal tubule cells, where it co-distributes with the Cl(-)/H(+) exchanger ClC-5 and Rab5a in endosomes. The biological relevance of CFTR in proximal tubule endocytosis has been tested in CF mouse models and CF patients. Mice lacking CFTR show a defective receptor-mediated endocytosis, as evidenced by impaired uptake of (125)I-beta(2)-microglobulin, a decreased expression of the cubilin receptor in the kidney, and a significant excretion of cubilin and its low-molecular-weight ligands into the urine. Low-molecular-weight proteinuria (and particularly transferrinuria) is similarly detected in CF patients in comparison with normal controls or patients with chronic lung inflammation. These studies suggest that the functional loss of CFTR impairs the handling of low-molecular-weight proteins by the kidney, supporting a role of CFTR in receptor-mediated endocytosis in proximal tubule cells. The selective proteinuria should be integrated in the pathophysiology of multi-systemic complications increasingly observed in CF patients.

ClC-2 participates in the regulation of neuronal excitability, chloride secretion, and cell volume. The ClC-2 sequence contains a consensus site (Ser82) for phosphorylation by the serum and glucocorticoid inducible kinase isoforms SGK1-3. Thus, the present study explored whether ClC-2 is regulated by those kinases. ClC-2 expression in Xenopus oocytes induced inwardly rectifying currents that increased upon coexpression of SGK1-3 and the related kinase PKB. The stimulatory effect was still present upon disruption of the SGK phosphorylation site. SGKs can phosphorylate the ubiquitin ligase Nedd4-2 and prevent Nedd4-2 from binding to its target. Therefore, the role of Nedd4-2 in ClC-2 modulation was investigated. ClC-2 activity decreased upon Nedd4-2 coexpression, an effect reversed by the kinases. According to chemiluminescence ClC-2 membrane abundance was enhanced by SGKs and diminished by Nedd4-2. These observations suggest that SGK1-3 and Nedd4-2 regulate ClC-2 at least in part by modulating ClC-2 abundance at the plasma membrane.

Alcaligenes eutrophus harboring plasmid pJP4 (strain JMP134) is capable of growing on both 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-Cba), while Pseudomonas putida carrying plasmid pAC27 (strain AC867) can utilize only 3-Cba as the sole carbon source. The tfdCDEF operon of the pJP4 plasmid and the clcABD operon of plasmid pAC27 each encode enzymes for the degradation of chlorocatechols (Clc), key intermediates in the catabolism of 2,4-D and 3-Cba. Similarities in the nucleotide (nt) sequences of genes tfdC and clcA, encoding pyrocatechases, were reported earlier [Ghosal and You, Mol. Gen. Genet. 211 (1988a) 113-120]. Genes tfdD and clcB, encoding Clc-specific cycloisomerases, have been completely sequenced. The tfdD gene (1107 bp) is slightly smaller than gene clcB (1113 bp). Comparison of the two cycloisomerase-encoding genes reveals that the nt sequences are 63% homologous with 62% homology in the deduced amino acid (aa) sequences of the polypeptides they encode. Genes tfdD and tfdE are contiguous in the tfdCDEF operon, whereas the corresponding genes, clcB and clcD, of the clcABD operon, are known to be separated by a long open reading frame of unknown function. The predicted N-terminal aa sequences of the two hydrolase-encoding genes, tfdE and clcD, also show homology. The structural and nt homologies between the two Clc operons, tfdCDEF and clcABD, suggest their relatedness.

Genes for metabolic pathways in bacteria that degrade aromatic or aliphatic pollutants have mostly been confined to either plasmid DNAs or to the chromosome. For a few pathways, including classical pathways for chlorocatechol and biphenyl degradation, recent evidence has been obtained for location of the pathway genes on mobile DNA elements which employ phage-like integrases. This enables the DNA elements to integrate into specific sites on the chromosome and yet to excise and transfer to other host bacteria. This mini-review gives an overview of those elements and their relationship to an increasing number of phage-like elements associated with bacterial virulence.

The fetal lung actively transports chloride across the airway epithelium. ClC-2, a pH-activated chloride channel, is highly expressed in the fetal lung and is located on the apical surface of the developing respiratory epithelium. Our goal was to determine whether acidic pH could stimulate chloride secretion in fetal rat distal lung epithelial cells mounted in Ussing chambers. A series of acidic solutions stimulated equivalent short-circuit current (I(eq)) from a baseline of 28 +/- 4.8 (pH 7.4) to 70 +/- 5 (pH 6.2), 114 +/- 12.8 (pH 5.0), and 164 +/- 19.2 (pH 3.8) microA/cm(2). These changes in I(eq) were inhibited by 1 mM cadmium chloride and did not result in large changes in [(3)H]mannitol paracellular flux. Immunofluorescent detection by confocal microscopy revealed that ClC-2 is expressed along the luminal surface of polarized fetal distal lung epithelial cells. These data suggest that the acidic environment of the fetal lung fluid could activate chloride channels contributing to fetal lung fluid production and that the changes in I(eq) seen in these Ussing studies may be due to stimulation of ClC-2.

In northern Finland myotonia congenita is caused by three main mutations in the ClC-1 chloride channel. We studied the molecular basis of these mutations (1238T>G/F413C, 1592C>T/A531V, and 2680C>T/R894X). The mutated cDNAs were expressed either in L6 myotubes or in isolated rat myofibers using recombinant Semliki Forest virus. Experiments in L6 cells indicated that A531V and R894X proteins suffered from stability problems in these cells. Analysis in myofibers indicated that the A531V protein was totally retained in the endoplasmic reticulum (ER), whereas the export of the F413C protein was severely reduced. The C-terminal nonsense mutant (R894X), however, was normally transported to the Golgi elements in the myofibers. Defective export or reduced stability of the mutated proteins may thus be reasons for the myotonic symptoms.

1. ClC-1 is a Cl- channel in mammalian skeletal muscle that plays an important role in membrane repolarization following muscular contraction. Reduction of ClC-1 conductance results in myotonia, a state characterized by muscle hyperexcitability. 2. As is the case for other members of the ClC family, ClC-1 exists as a dimer that forms a double-barrelled channel. Each barrel, or pore, of ClC-1 is gated by its own gate ('fast' or 'single pore' gate), whereas both pores are gated simultaneously by another mechanism ('slow' or 'common' gate). 3. Comparison of the biophysical and pharmacological properties of heterologously expressed ClC-1 with the properties of the Cl- conductance measured in skeletal muscle strongly suggests that ClC-1 is the major Cl- channel responsible for muscle repolarization. However, not all results obtained in experiments on whole muscle or muscle fibres support this notion. 4. In the present review we attempt to bring together the current knowledge of ClC-1 with the physiology of skeletal muscle.

Thirty-nine cases of pulmonary paragonimiasis due to Paragonimus westermani were identified in Manipur, India. Recurrent haemoptysis, often increased on exertion, was the most common symptom. The clinical features and radiological appearances simulated pulmonary tuberculosis and 23 cases were treated for tuberculosis for varying periods before the final diagnosis. Young males aged 11 to 30 years comprised two-thirds of the cases. The disease was acquired by eating raw or improperly cooked crabs (Potamon deham). Intradermal tests (IDT) employing a saline extract of adult Paragonimus were a simple and reliable method of detecting present or past infection. Erythema of large size even without wheal and pseudopodia may indicate a positive IDT. Charcot-Leyden crystals (CLC) were detected in all sputum samples positive for Paragonimus ova. In patients (four) presenting with pleural effusion, Paragonimus ova were not present in the sputum, stool or pleural fluid. Bithionol (2,2'-thiobis [4,6-dichlorophenol]) orally, 40 mg/kg body-weight/day, 10 to 25 doses, cured all cases and, except in one, side effects were minimal. Relapse in one patient after six months' treatment was apparently cured by a second course of Bithionol.

BACKGROUND

Antipsychotic medications are commonly prescribed to nursing home residents despite their well-established adverse event profiles. Because little is known about their use in Veterans Affairs (VA) nursing homes [ie, Community Living Centers (CLCs)], we assessed the prevalence and risk factors for antipsychotic use in older residents of VA CLCs.

METHODS

This cross-sectional study included 3692 Veterans age 65 or older who were admitted between January 2004 and June 2005 to one of 133 VA CLCs and had a stay of ≥90 days. We used VA Pharmacy Benefits Management data to examine antipsychotic use and VA Medical SAS datasets and the Minimum Data Set to identify evidence-based indications for antipsychotic use (eg, schizophrenia, dementia with psychosis). We used multivariable logistic regression and generalized estimating equations to identify factors independently associated with antipsychotic receipt.

RESULTS

Overall, 948/3692(25.7%) residents received an antipsychotic, of which 59.3% had an evidence-based indication for use. Residents with aggressive behavior [odds ratio (OR)=2.74, 95% confidence interval (CI), 2.04-3.67] and polypharmacy (9+ drugs; OR=1.84, 95% CI, 1.41-2.40) were more likely to receive antipsychotics, as were users of antidepressants (OR=1.37, 95% CI, 1.14-1.66), anxiolytic/hypnotics (OR=2.30, 95% CI, 1.64-3.23), or drugs for dementia (OR=1.52, 95% CI, 1.21-1.92). Those residing in Alzheimer/dementia special care units were also more likely to receive an antipsychotic (OR=1.66, 95% CI, 1.26-2.21). Veterans with dementia but no documented psychosis were as likely as those with an evidence-based indication to receive an antipsychotic (OR=1.10, 95% CI, 0.82-1.47).

CONCLUSIONS

Antipsychotic use is common among VA nursing home residents aged 65 and older, including those without a documented evidence-based indication for use. Further quality improvement efforts are needed to reduce potentially inappropriate antipsychotic prescribing.

BACKGROUND

Disorders that manifest hypokalemic metabolic alkalosis, such as Bartter's syndrome and Gitelman's syndrome, are caused by the malfunction of renal tubular electrolyte transporters. Bartter's syndrome may be linked to dysfunction of Na-K-2Cl cotransporter (NKCC2), renal outer medullary K channel (ROMK), or Cl channel Kb (ClC-Kb), while Gitelman's syndrome may be linked to Na-Cl cotransporter (NCCT) dysfunction. However, previous genetic analyses in these syndromes have included many heterozygotes for each gene and there has been no further analysis of other genes. Thus, to clarify the interaction of these transporter genes, in the present study we investigated all 4 transporter genes in 7 patients with hypokalemic metabolic alkalosis.

METHODS

Seven patients from 5 families (patients A-G) were collected, and a mutation analysis of the 4 renal electrolyte transporter genes was performed by direct sequencing.

RESULTS

We identified 12 mutations in these 7 patients. Three mutations (del245Y in NKCC2, R1009X in NCCT, V524I in ClC-Kb) have not been reported previously. In NKCC2 gene screening, patient A was homozygous for del245Y. In ClC-Kb gene screening, L27R was detected in patients B, D, and E. V524I was detected in patient C. Both T562M and E578K were observed in patients B and E. In NCCT gene screening, patients B-G shared a common novel mutation, R1009X, and patients D, E, F, and G carried this mutation in both alleles. Patients B and C carried R1009X in one allele, and a 6-amino acid insertion in exon 6 and L849H in another allele, respectively. The 4 other mutations did not result in any amino acid exchange. Despite the NCCT gene mutation, patients C and E showed normomagnesemia.

CONCLUSIONS

Our findings demonstrate that in Bartter's and Gitelman's syndromes, it may not be uncommon to see mutations in several causative transporter genes.

The molecular identification of cardiac chloride channels has provided probes to investigate their distribution and abundance in heart. In this study, the molecular expression and distribution of volume-regulated chloride channels ClC-2 and ClC-3 in cardiac tissues were analyzed and quantified. Total RNA was isolated from atria and ventricles of several species (dog, guinea pig, and rat) and subjected to a quantitative RT-PCR strategy. ClC-2 and ClC-3 mRNA expression were calculated relative to beta-actin expression within these same tissues. The transcriptional levels of ClC-3 mRNA were between 1.8 and 10.2% of beta-actin expression in atria and between 3.4 and 8.6% of beta-actin in ventricles (n = 3 for each tissue). The levels of ClC-2 in both atria and ventricles were significantly less than those measured for ClC-3 (n = 3; P < 0.05). ClC-2 mRNA levels were between 0.04-0.08% and 0.03-0.18% of beta-actin expression in atria and ventricles, respectively (n = 3 for each tissue). Immunoblots of atrial and ventricular wall protein extracts demonstrated ClC-2- and ClC-3-specific immunoreactivity at 97 and 85 kDa, respectively. Immunohistochemical localization in guinea pig cardiac muscle demonstrates a ubiquitous distribution of ClC-2 and ClC-3 channels in the atrial and ventricular wall. Confocal analysis detected colocalization of ClC-2 and ClC-3 in sarcolemmal membranes and distinct ClC-3 immunoreactivity in cytoplasmic regions. The molecular expression of ClC-2 and ClC-3 in cardiac tissue is consistent with the proposed role of these chloride channels in the regulation of cardiac cell volume and the modulation of cardiac electrical activity.

Several Cl(-) channels have been described in the native renal tubule, but their correspondence with ClC-K1 and ClC-K2 channels (orthologs of human ClC-Ka and ClC-Kb), which play a major role in transcellular Cl(-) absorption in the kidney, has yet to be established. This is partly because investigation of heterologous expression has involved rat or human ClC-K models, whereas characterization of the native renal tubule has been done in mice. Here, we investigate the electrophysiological properties of mouse ClC-K1 channels heterologously expressed in Xenopus laevis oocytes and in HEK293 cells with or without their accessory Barttin subunit. Current amplitudes and plasma membrane insertion of mouse ClC-K1 were enhanced by Barttin. External basic pH or elevated calcium stimulated currents followed the anion permeability sequence Cl(->>Br(->>NO3(->>I(-). Single-channel recordings revealed a unit conductance of ~40pS. Channel activity in cell-attached patches increased with membrane depolarization (voltage for half-maximal activation: ~-65mV). Insertion of the V166E mutation, which introduces a glutamate in mouse ClC-K1, which is crucial for channel gating, reduced the unit conductance to ~20pS. This mutation shifted the depolarizing voltage for half-maximal channel activation to ~+25mV. The unit conductance and voltage dependence of wild-type and V166E ClC-K1 were not affected by Barttin. Owing to their strikingly similar properties, we propose that the ClC-K1/Barttin complex is the molecular substrate of a chloride channel previously detected in the mouse thick ascending limb (Paulais et al., J Membr. Biol, 1990, 113:253-260).

Chloride (Cl) ion channels play a critical role in the response of both vascular smooth muscle (VSM) and endothelial (ENDO) cells to agonist stimulation. In VSM, agonist-induced Cl currents produce membrane depolarization, resulting in calcium influx through voltage-sensitive channels. ENDO cells also activate Cl currents after either agonist application or perturbation of cell volume. Although some of these currents have been characterized biophysically, the genes involved have not been identified. The CLCN family of voltage-dependent Cl channel genes comprises nine members (CLCN1-7, Ka and Kb) which demonstrate quite diverse functional characteristics while sharing significant sequence homology. We used Northern-blot analysis to study the expression of these Cl channel genes in cultured human aortic and coronary VSM cells and in aortic ENDO cells. CLCN3 is by far the most abundant CLC channel mRNA in both VSM and ENDO cells. Lower levels of expression are seen for CLCN2, CLCN4, CLCN5 and CLCN6. Expression levels were similar in VSM and ENDO cells except for CLCN4 which was more highly expressed in ENDO cells. In situ hybridization was used to confirm the expression of CLCN3 in intact human fetal lung. CLCN3 message was seen in VSM and ENDO cells of both large and small pulmonary vessels, indicating that their detection by Northern blotting was not an artifact of cell culture. CLCN3 is also expressed in pulmonary epithelial and bronchial smooth muscle cells but not in chondrocytes or pulmonary interstitial cells. Recent studies suggest that CLCN3 may encode the swelling-induced Cl conductance. We used whole cell patch clamp recording to demonstrate swelling-induced Cl currents in these cultured VSM cells. This suggests that the CLCN3 protein is expressed; however, the functional role of this current in VSM remains to be determined.

Six <em>ClC</em>-type chloride channel genes have been identified in Caenorhabditis elegans, termed clh-1 through clh-6. cDNA sequences from these genes suggest that clh-2, clh-3, and clh-4 may code for multiple channel variants, bringing the total to at least nine channel types in this nematode. Promoter-driven green fluorescent protein (GFP) expression in transgenic animals indicates that the protein CLH-5 is expressed ubiquitously, CLH-6 is expressed mainly in nonneuronal cells, and the remaining isoforms vary from those restricted to a single cell to those expressed in over a dozen cells of the nematode. In an Sf9 cell expression system, recombinant CLH-2b, CLH-4b, and CLH-5 did not form functional plasma membrane channels. In contrast, both CLH-1 and CLH-3b produced strong, inward-rectifying chloride currents similar to those arising from mammalian <em>ClC</em>2, but which operate over different voltage ranges. Our demonstration of multiple CLH protein variants and comparison of expression patterns among the clh gene family provides a framework, in combination with the electrical properties of the recombinant channels, to further examine the physiology and cell-specific role each isoform plays in this simple model system.

Lymphoid cells obtained from the spleen, thymus, bone marrow, peripheral blood, and peritoneal exudate of normal mice (BALB/c, BALB/c nude, C57BL/6, C3H) and from spleens of mice bearing a transplantable lung carcinoma or primary mammary carcinoma were expanded in culture for 1-9 months, with an increase in cell number of 10(5)- to 10(6)-fold per month, in crude or lectin-depleted medium containing T cell growth factor (TCGF). All these cultured lymphoid cell (CLC) lines exhibited strong cytotoxic activity in vitro (assessed by 51Cr-release assays) toward a variety of freshly harvested and cultured syngeneic, allogeneic, and xenogeneic tumor target cells, both lymphoid and solid (including metastatic growths) in origin. Extensive killing was observed against tumor targets that were resistant to lysis by natural killer (NK) cells as well as to NK-sensitive tumor lines. Low levels of cytotoxic reactivity were also demonstrated against fresh and cultured normal lymphoid cells. The CLC had some characteristics of NK cells but also expressed some typical T cell markers. In local Winn-type neutralization assays, CLC delayed or completely inhibited the growth of lymphomas and carcinomas in syngeneic and allogeneic recipients. In mice with metastatic growth of a second-generation transplant of mammary carcinoma, CLC were shown to have some therapeutic effect when administered IV 1 day after cyclophosphamide. No significant beneficial action of IV administered CLC was observed in the absence of chemotherapy in mice implanted with a lung carcinoma. The possibilities of employing TCGF-propagated cytotoxic effector cells in adoptive immunotherapy of human malignancies are discussed.

We describe the isolation and characterization of a human gene (CLCN3) and its murine homologue (Clcn3) sharing significant sequence and structural similarities with all previously identified members of the voltage-gated chloride channel (ClC) family. This gene is expressed primarily in tissues derived from neuroectoderm. Within the brain, Clcn3 expression is particularly evident in the hippocampus, olfactory cortex, and olfactory bulb. CLCN3 encodes a 760-amino-acid protein that differs by only 2 amino acid residues from the protein encoded by Clcn3. CLCN3 protein also shows a high similarity with GEF1, an integral membrane protein of the yeast Saccharomyces cerevisiae known to be involved in respiration and iron-limited cell growth, and with the predicted protein product of a DNA sequence from the mold Septoria nodorum. This high degree of sequence conservation in very distantly related species such as human and yeast indicates that this gene has retained a fundamental function throughout evolution.

The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na+-Cl- cotransporter located at the apical membrane, and Na+, K+ ATPase at the basolateral side. Cl- transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl- channels on microdissected mouse DCTs using the patch-clamp technique. A channel of approximately 9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NPo in cell-attached patches was not modified when tubules were preincubated in the presence of 10-5 M forskolin, but the channel was inhibited by phorbol ester (10-6 M). In addition, NPo was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM (NPo increased threefold), or pH increased from 6.4 to 8.0 (NPo increased 15-fold). Selectivity experiments conducted on inside-out patches showed that the Na+ to Cl- relative permeability was 0.09, and the anion selectivity sequence Cl(-)--I(-)>> Br(-)--NO3(-)>> F(-). Intracellular NPPB (10-4 M) and DPC (10-3 M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl- channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family.

Defolliculated oocytes of Xenopus laevis responded to removal of external divalent cations with large depolarizations and, when voltage clamped, with huge currents. Single channel analysis revealed a Cl- channel with a slope conductance of about 90 pS at positive membrane potentials with at least four substates. Single channel amplitudes and mean channel currents had a reversal potential of approximately -15 mV as predicted by the Nernst equation for a channel perfectly selective for Cl-. Readdition of Ca2+ immediately inactivated the channel and restored the former membrane potential or clamp current. The inward currents were mediated by a Ca2+ inactivated Cl- channel (CaIC). The inhibitory potency of Ca2+ was a function of the external Ca2+ concentration with a half maximal blocker concentration of about 20 microM. These channels were inhibited by the Cl- channel blockers flufenamic acid, niflumic acid and diphenylamine-2-carboxylate (DPC). In contrast, 4,4'-acetamido-4'-isothiocyanatostilbene-2, 2'-disulfonicacid (SITS), another Cl- channel blocker, led to activation of this Cl- channel. Like other Cl- channels, the CaIC was activated by cytosolic cAMP. Extracellular ATP inhibited the channel while ADP was without any effect. Injection of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activating phorbol ester, stimulated the Cl- current. Cytochalasin D, an actin filament disrupting compound, reversibly decreased the clamp current demonstrating an influence of the cytoskeleton. The results indicate that removal of divalent cations activates Cl- channels in Xenopus oocytes which share several features with Cl- channels of the CLC family. The former so-called leak current of oocytes under divalent cation-free conditions is nothing else than an activation of Cl- channels.

Aquaporins (AQP) are involved in rapid and active gating of water across biological membranes. The molecular regulation of AQP is unknown. Here we report the isolation, identification and reconstitution of the regulatory complex of AQP-1. AQP-1 and Galphai3 have been implicated in GTP-induced gating of water in zymogen granules (ZG), the secretory vesicles in exocrine pancreas. In the present study, detergent-solubilized ZGs immunoprecipitated with monoclonal AQP-1 antibody, co-isolates AQP-1, PLA2, Galphai3, potassium channel IRK-8, and the chloride channel ClC-2. Exposure of ZGs to either the potassium channel blocker glyburide, or the PLA2 inhibitor ONO-RS-082, blocked GTP-induced ZG swelling. RBC known to possess AQP-1 at the plasma membrane, swell on exposure to the Galphai-agonist mastoparan, and respond similarly to ONO-RS-082 and glyburide, as ZGs. Liposomes reconstituted with the AQP-1 immunoisolated complex from solubilized ZG, also swell in response to GTP. Glyburide or ONO-RS-082 abolished the GTP effect. Immunoisolate-reconstituted planar lipid bilayers demonstrate conductance, which is sensitive to glyburide and an AQP-1 specific antibody. Our results demonstrate a Galphai3-PLA2 mediated pathway and potassium channel involvement in AQP-1 regulation.