CLC-K Cl(-) channels belong to the CLC protein family. In kidney and inner ear, they are involved in transepithelial salt transport. Mutations in ClC-Kb lead to Bartter's syndrome, and mutations in the associated subunit barttin produce Bartter's syndrome and deafness. We have previously found that 3-phenyl-CPP blocks hClC-Ka and rClC-K1 from the extracellular side in the pore entrance. Recently, we have shown that niflumic acid (NFA), a nonsteroidal anti-inflammatory fenamate, produces biphasic behavior on human CLC-K channels that suggests the presence of two functionally different binding sites: an activating site and a blocking site. Here, we investigate in more detail the interaction of NFA on CLC-K channels. Mutants that altered block by 3-phenyl-2-(p-chlorophenoxy)propionic acid (CPP) had no effect on NFA block, indicating that the inhibition binding site of NFA is different from that of 3-phenyl-CPP and flufenamic acid. Moreover, NFA does not compete with extracellular Cl(-) ions, suggesting that the binding sites of NFA are not located deep in the pore. Differently from ClC-Ka, on the rat homologue ClC-K1, NFA has only an inhibitory effect. We developed a quantitative model to describe the complex action of NFA on ClC-Ka. The model predicts that ClC-Ka possesses two NFA binding sites: when only one site is occupied, NFA increases ClC-Ka currents, whereas the occupation of both binding sites leads to channel block.

Studies on rats have shown that lactic acid can improve excitability and function of depolarized muscles. The effect has been related to the ensuing reduction in intracellular pH causing inhibition of muscle fibre Cl(-) channels. However, since several carboxylic acids with structural similarities to lactate can inhibit muscle Cl(-) channels it is possible that lactate per se can increase muscle excitability by exerting a direct effect on these channels. We therefore examined the effects of lactate on the function of intact muscles and skinned fibres together with effects on pH and Cl(-) conductance (G(cl)). In muscles where extracellular compound action potentials (M-waves) and tetanic force response to excitation were reduced by (mean ± s.e.m.) 82 ± 4% and 83 ± 2%, respectively, by depolarization with 11 mm extracellular K(+), both M-waves and force exhibited an up to 4-fold increase when 20 mm lactate was added. This effect was present already at 5 mm and saturated at 15 mm lactate, and was associated with a 31% reduction in G(Cl). The effects of lactate were completely blocked by Cl(-) channel inhibition or use of Cl(-)-free solutions. Finally, both experiments where effects of lactate on intracellular pH in intact muscles were mimicked by increased CO₂ tension and experiments with skinned fibres showed that the effects of lactate could not be related to reduced intracellular pH. It is concluded that addition of lactate can inhibit ClC-1 Cl(-) channels and increase the excitability and contractile function of depolarized rat muscles via mechanisms not related to a reduction in intracellular pH.

OBJECTIVE

Hyposmolar perfusion of intact trabecular meshwork (TM) induces a decrease in its hydraulic conductivity (Lp). However, exposure to agents that elevate intracellular cAMP in TM cells increases Lp. Since volume of TM cells could directly influence porosity of the TM and hence Lp, this study has investigated changes in volume in response to acute hyposmotic shock (i.e. regulatory volume decrease or RVD) and elevated cAMP in cultured TM cells.

METHODS

Bovine trabecular meshwork cells (BTMC), grown on glass coverslips and loaded with the fluorescent dye MQAE, were used to measure rapid changes in cell volume using the principle of dynamic fluorescence quenching. Activation of volume-regulated anion channels (VRAC) was assessed by measuring volume-sensitive Cl(-) currents (I(Cl,swell)) in the whole cell configuration of the patch clamp technique and by determining the swelling-induced enhancement in I(-) permeability using the halide-sensitivity of MQAE. Expressions of ClC (chloride channels of the ClC gene family), P-glycoprotein (Pgp), and cystic fibrosis transmembrane regulator (CFTR) Cl(-) channels were examined by RT-PCR. Elevation of cAMP in response to forskolin was confirmed by determining the phosphorylation of cAMP response element-binding protein and activating transcription factor-1 (CREB, ATF-1), which form the downstream targets of protein kinase A.

RESULTS

As a response to hyposmotic shock, there was an acute increase in cell volume but there was no robust RVD. Patch clamp experiments showed activation of a characteristic Cl(-) current in response to cell swelling. This Cl(-) current was inhibited by NPPB (100microM) and fluoxetine (50microM), both of which are known blockers of VRAC. Experiments, which used the halide-sensitivity of MQAE, also indicated a 9-fold increase in I(-) influx upon cell swelling (8.9+/-4.6; n=9), consistent with activation of a VRAC-like Cl(-) current. To examine whether RVD is limited by K(+) conductance, the swollen cells were exposed to gramicidin, which is known to induce cation channel activity. Such a maneuver led to secondary swelling with [Na(+)](o)=140mM but a rapid shrinkage [Na(+)](o)=8mM indicating that the RVD is limited by cationic conductance necessary for K(+) efflux. Exposure to forskolin, which resulted in CREB and ATF-1 phosphorylation, caused a reversible decrease in cell volume (14.5+/-5%; n=20) under isosmotic and hyposmotic conditions. RT-PCR analysis confirmed expression of ClC-2, ClC-5, and Pgp Cl(-) channels in bovine TM cells. However, ClC-3 and CFTR were not expressed.

CONCLUSIONS

TM cells respond to acute hyposmotic shock in an osmometric manner, but their RVD is limited by K(+) conductance. The lack of CFTR expression and decrease in cell volume in response to forskolin concomitant with hyposmolarity suggest that elevated cAMP activates a K(+) conductance. Thus, the altered resistance to aqueous outflow in response to hyposmotic perfusion of the TM and elevated cAMP may be attributed to persistent cell swelling and cell shrinkage, respectively.

Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A2 (cPLA2alpha) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca2+-dependent exocytosis in single mouse pancreatic beta-cells. cPLA2alpha dose dependently (EC50 = 86 nM) stimulated depolarization-evoked exocytosis by 450% without affecting the whole cell Ca2+ current or cytoplasmic Ca2+ levels. The stimulatory effect involved priming of secretory granules as reflected by an increase in the size of the readily releasable pool of granules from 70-80 to 280-300. cPLA2alpha-stimulated exocytosis was antagonized by the specific cPLA2 inhibitor AACOCF3. Ca2+-evoked exocytosis was reduced by 40% in cells treated with AACOCF3 or an antisense oligonucleotide against cPLA2alpha. The action of cPLA2alpha was mimicked by a combination of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl- uptake through ClC-3 Cl- channels. Accordingly, the stimulatory action of cPLA2alpha was inhibited by the Cl- channel inhibitor DIDS and in cells pretreated with ClC-3 Cl- channel antisense oligonucleotides. We propose that cPLA2alpha has an important role in controlling the rate of exocytosis in beta-cells. This effect of cPLA2alpha reflects an enhanced transgranular Cl- flux, leading to an increase in the number of granules available for release, and requires the combined actions of arachidonic acid and lysophosphatidylcholine.

Rat parotid acinar cells express Cl- currents that are activated in a time-dependent manner by hyperpolarized potentials. ClC-2, a member of the ClC gene family, codes for a voltage-gated, inward rectifying anion channel when expressed in Xenopus oocytes. In the present study, we found that cDNA derived from individual parotid acinar cells contained sequence identical to that reported for ClC-2 in rat brain and heart. A polyclonal antibody generated against the N-terminal cytoplasmic domain of ClC-2 recognized an approximately 100 kD protein on western blots of both brain and parotid gland. ClC-2 expressed in oocytes has different kinetics from the currents found in parotid acinar cells. Since the ClC-2 channel was cloned from and its transcripts are expressed in mammalian tissue, we compared the channel properties of acinar cells to a mammalian expression system. We expressed ClC-2 channels in human embryonic kidney cells, HEK 293, using recombinant ClC-2 DNA and ClC-2 DNA fused with DNA coding for jellyfish green fluorescent protein (GFP). Confocal microscopy revealed that the expressed ClC-2-GFP chimera protein localized to the plasma membrane. Whole cell Cl- currents from HEK 293 cells expressing ClC-2-GFP were similar, if not identical, to the Cl- currents recorded from cells transfected with ClC-2 cDNA (no GFP). The voltage-dependence and kinetics of ClC-2 channels expressed in HEK 293 cells were quite similar to those in acinar cells. Channels in parotid acinar and HEK 293 cells activated at more positive membrane potentials and with a faster time course than the channels expressed in Xenopus oocytes. In summary, we found that ClC-2 message and protein are expressed in salivary cells and that the properties of voltage-activated, inward rectifying Cl- channels in acinar cells are similar to those generated by the ClC-2-GFP construct expressed in HEK 293 cells. The properties of the ClC-2 anion channel seem to be dependent on the type of cell background in which it is expressed.

Hypokalemic metabolic tubulopathy, such as in Bartter syndrome and Gitelman syndrome, is caused by the dysfunction of renal electrolyte transporters. Despite advances in molecular genetics with regard to hypokalemic metabolic tubulopathy, recent reports have suggested that the phenotype-genotype correlation is still confusing, especially in classic Bartter and Gitelman syndromes. We report here two Japanese patients who suffered from clinically diagnosed classic Bartter syndrome but who presented hypocalciuria. Hypocalciuria is generally believed to be a pathognomonic finding of NCCT malfunction. To better understand the genotype-phenotype correlation in these two cases, we screened four renal electrolyte transporter genes [Na-K-2Cl cotransporter (NKCC2), renal outer medullary K channel (ROMK), Cl channel Kb (ClC-Kb), and Na-Cl cotransporter (NCCT)] by the PCR direct sequencing method. We identified three ClC-Kb allelic variants, including two new mutations (L27R and W610X in patient 1 and a G to C substitution of a 3' splice site of intron 2 and W610X in patient 2). We did not find any mutations in the other three genes. Our present data suggest that some ClC-Kb mutations may affect calcium handling in renal tubular cells.

Despite considerable similarity in their amino acid sequences and structural features, the mammalian members of the CLC chloride channel/transporter family have different subcellular locations. The subcellular location and function of one of these members, hClC-4, is controversial. To characterize its cellular function, we investigated its tissue distribution and subcellular location. Expression was high in excitable tissues such as the nervous system and skeletal muscle. When heterologously expressed in HEK293 cells and in skeletal muscle fibers, hClC-4 localizes to the endoplasmic/sarcoplasmic reticulum (ER/SR) membranes, in contrast to hClC-3, which localizes to vesicular structures. This location was confirmed by identification of endogenous ClC-4 in membrane fractions from mouse brain homogenate enriched for the sarco-endoplasmic reticulum ATPase SERCA2, an ER/SR marker. To identify the motif responsible for ER localization of hClC-4, we generated hClC-4 truncations and chimeras between hClC-4 and hClC-3 or the unrelated plasma membrane protein Ly49E. A stretch of amino acids, residues 14-63, at the N-terminus constitutes a novel motif both necessary and sufficient for targeting hClC-4 and other membrane proteins to the ER.

OBJECTIVE

Recent findings relevant to the renal ClC chloride channels/transporters are reviewed with a focus on structure-function relationships, regulation of trafficking, role in blood pressure control, and pharmacology.

RESULTS

The ClC proteins include plasma membrane Cl channels and vesicular Cl/H exchangers. Recent experiments have revealed further details regarding the structure and mechanism of the permeation path. X-ray crystallographic and electrophysiological studies have identified two glutamate residues required for gated Cl movement and proton permeation in bacterial and two mammalian (ClC-4, ClC-5) ClC transporters. In renal ClC channels (ClC-Ka, ClC-Kb), both glutamate residues are replaced by valine, leading to speculation about critical differences between transporter and channel members of the ClC family. New information about the physiological regulation of renal ClC proteins has implicated the Nedd4 ubiquitin ligases and serum and glucocorticoid-inducible kinases in controlling functional levels of ClC-5 and ClC-K/barttin in renal cells.

CONCLUSIONS

ClC proteins are critical for many clinically relevant physiological events. New insights into fundamental structure-function relationships, mechanisms of ion translocation, cellular regulation, and roles in human disease have increased attention on ClC proteins as important potential therapeutic targets.

We have previously shown that mouse mandibular granular ducts contain a hyperpolarization-activated Cl- conductance. We now show that the instantaneous current/voltage (I/V) relation of this Cl- conductance is inwardly rectifying with a slope conductance of 15.4 +/- 1.8 nS (n = 4) at negative potentials and of 6.7 +/- 0.9 nS (n = 4) at positive potentials. Thus, the inward rectification seen in the steady-state I/V relation is due, not only to voltage activation of the Cl- conductance, but also to the intrinsic conductance properties of the channel. We show further that the ductal Cl- conductance is not activated by including ATP (10 mmol/l) in the pipette solution. Finally, we show that the conductance is not blocked by the addition of any of the following compounds to the extracellular solution: anthracene-9-carboxylate (A9C, 1 mmol/l), diphenylamine-2-carboxylate (DPC, 1 mmol/l), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 mumol/l), 4,4'-diisothiocyanato-stilbene-2,2'-disulphonate (DIDS, 100 mumol/l), indanyloxyacetic acid (IAA-94, 100 mumol/l), verapamil (100 mumol/l), glibenclamide (100 mumol/l) and Ba2+ (5 mmol/l). The properties of the ductal Cl- conductance most nearly resemble those of the ClC-2 channel. Both channel types have instantaneous I/V relations that are slightly inwardly rectifying, are activated by hyperpolarization with a time-course in the order of hundreds of milliseconds, have a selectivity sequence of Br->> Cl->> I-, and are insensitive to DIDS. The only identified difference between the two is that the ClC-2 channel is 50% blocked both by DPC and A9C (1 mmol/l), whereas the ductal Cl- conductance is insensitive to these compounds.

In Pseudomonas putida, benzoate and 3-chlorobenzoate are converted to catechol and 3-chlorocatechol, respectively, which are then catabolized to tricarboxylic acid cycle intermediates via the catBCA and clcABD pathways. The catBCA and clcABD operons are regulated by homologous transcriptional activators CatR and ClcR. Previous studies have demonstrated that in addition to sequence similarities, CatR and ClcR share functional similarities which allow catR to complement clcR. In this study, we demonstrate that CatR activates the clcABD promoter in vitro without inducer, but more transcript is produced when inducer is added. DNase I footprinting and DNA-bending analyses demonstrate that CatR binds to and bends the clcABD promoter to the same angle as does ClcR plus its inducer, 2-chloromuconate. This implies that CatR binds to the clc promoter in its active conformation. Transcription of the clcABD promoter by the alpha-subunit truncation mutant (alpha-235) of RNA polymerase was sharply reduced, indicating that the alpha-subunit C-terminal domain is important. However, a small amount of transcript was produced under these conditions, indicating that other contact sites on the RNA polymerase may play a role in activation.

It is well established that granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1 and tumour necrosis factor-alpha (TNF-alpha) are involved in Langerhans' cell (LC) development and dendritic cell traffic. However, little is known about the pattern of cytokine receptors on human LC and their modulation during different stages of maturation. The expression of cytokine receptors was studied by flow cytometry on both freshly isolated LC (fLC) and 72-hr cultured LC (cLC). Epidermal cell suspensions enriched in LC were obtained after skin trypsinization and Ficoll-Hypaque gradient. LC were identified by their CD1a positivity. Although the majority of fLC were positive for the alpha chain of GM-CSF receptor (GM-CSFR), the beta chain of GM-CSFR was detected only on 15% of CD1a+ cells. fLC were also positive for IL-1 receptor (IL-1R) type 1, IL-1R type 2, 75,000 molecular weight TNF receptor (TNFR) and interferon-gamma receptor (IFN-gamma R). IL-6R and its transducing signal gp130 were present in a subset of fLC. Granulocyte colony-stimulating factor receptor (G-CSFR), macrophage colony-stimulating factor receptor (M-CSFR), the alpha and beta chain of IL-2R, IL-4R, IL-7R, IL-8R and 55,000 molecular weight TNFR were not detected on fLC. After culture, LC up-regulated the expression of both the alpha and beta chains of GM-CSFR, IL-1R type 2, alpha and beta chains of IL-2R, IL-6R and gp130. In contrast, IL-1R type 1 and 75,000 molecular weight TNFR were down-modulated and the expression of IFN-gamma R was not affected by culture. These results suggest that LC undergo changes in the cytokine receptor repertory during in vitro maturation.

BACKGROUND

Studies of closed-loop control (CLC) systems have improved glucose levels in patients with type 1 diabetes. In this study we test a new CLC concept aiming to "reset" the patient overnight to near-normoglycemia each morning, for several consecutive nights.

METHODS

Ten insulin pump users with type 1 diabetes (mean age, 46.4±8.5 years) were enrolled in a two-center (in the United States and Italy) randomized crossover trial comparing 5 consecutive nights of CLC (23:00-07:00 h) in an outpatient setting versus sensor-augmented insulin pump therapy of the same duration at home. Primary end points included time spent in 80-140 mg/dL as measured by continuous glucose monitoring overnight and fasting blood glucose distribution at 7:00 h.

RESULTS

Compared with sensor-augmented pump therapy, CLC improved significantly time spent between 80 and 140 mg/dL (54.5% vs. 32.2%; P<0.001) and between 70 and 180 mg/dL (85.4% vs. 59.1%; P<0.001); CLC reduced the mean glucose level at 07:00 h (119.3 vs. 152.9 mg/dL; P<0.001) and overnight mean glucose level (139.0 vs. 170.3 mg/dL; P<0.001) using a marginally lower amount of insulin (6.1 vs. 6.8 units; P=0.1). Tighter overnight control led to improved daytime control on the next day: the overnight/next-day control correlation was r=0.52, P<0.01.

CONCLUSIONS

Multinight CLC of insulin delivery (artificial pancreas) results in significant improvement in morning and overnight glucose levels and time in target range, with the potential to improve daytime control when glucose levels were "reset" to near-normoglycemia each morning.

BACKGROUND

Studies of the nasal lavage fluid proteome have previously identified proteins differently expressed in patients with symptomatic allergic rhinitis, e.g. S100A7, prolactin-inducible protein (PIP), wingless-type MMTV integration site family, member 2B (WNT2B), Charcot-Leyden crystal protein (CLC) and palate lung nasal epithelial clone (PLUNC). The aim of the present study was to investigate if genetic variation associated with allergic rhinitis can be found in these genes.

METHODS

Peripheral blood was collected from 251 patients with birch and/or grass pollen-induced allergic rhinitis and 386 nonatopic healthy controls. A total of 39 single nucleotide polymorphisms (SNPs) distributed over the genes PIP, WNT2B, CLC and PLUNC were selected from dbSNP, genotyped and investigated for associations with allergic rhinitis. Twelve additional SNPs were subsequently analysed for CLC.

RESULTS

All 22 investigated SNPs in CLC were polymorphic. Ten SNPs yielded significant differences between cases and controls with respect to genotype frequencies. Homozygotes for the minor allele were more common in allergic individuals compared to healthy controls. The minor alleles of these SNPs were all located on the same haplotype. Furthermore, homozygotes for the minor allele of two of the promoter SNPs had higher average scores for birch in skin prick test. In contrast, for seven SNPs within the gene, heterozygotes and homozygotes for the major allele had higher average scores for grass. None of the other three genes showed association.

CONCLUSIONS

Genetic variation in CLC was found to be associated with allergic rhinitis. The pattern of variation is compatible with a recessive inheritance model and the previously observed altered protein levels detected in patients with allergic rhinitis.

It has been difficult to separate/identify the roles of ClC-2 and CFTR in Cl(-) transport studies. Using pharmacological agents, we aimed to differentiate functionally between ClC-2 and CFTR Cl(-) channel currents. Effects of CFTR inhibitor 172 (CFTRinh172), N-(4-methylphenylsulfonyl)-N'-(4-trifluoromethylphenyl)urea (DASU-02), and methadone were examined by whole cell patch clamp on Cl(-) currents in recombinant human ClC-2/human embryonic kidney 293 (ClC-2/HEK293) cells stably transformed with Epstein-Barr nuclear antigen 1 (hClC-2/293EBNA) and human CFTR/HEK293 (hCFTR/HEK293) cells and by short-circuit current (Isc) measurements in T84 cells. Lubiprostone and forskolin-IBMX were used as activators. CFTRinh172 inhibited forskolin-IBMX-stimulated recombinant human CFTR (hCFTR) and lubiprostone-stimulated recombinant human ClC-2 (hClC-2) Cl(-) currents in a concentration-dependent manner equipotently. DASU-02 inhibited forskolin-IBMX-stimulated Cl(-) currents in hCFTR/HEK293 cells, but not lubiprostone-stimulated Cl(-) currents in hClC-2/293EBNA cells. In T84 cells with basolateral nystatin or 1-ethyl-2-benzimidazolinone (1-EBIO), lubiprostone-stimulated and forskolin-IBMX-cyclosporin A (FICA)-stimulated Isc components were observed. CFTRinh172 inhibited major portions of both components. DASU-02 had no effect on lubiprostone-stimulated Isc but partially inhibited FICA-stimulated Isc. T84 cells in which ClC-2 or CFTR was knocked down using siRNAs were constructed. T84 ClC-2 knockdown cells did not respond to lubiprostone but did respond to forskolin-IBMX in a methadone-insensitive, DASU-02-sensitive manner, indicating CFTR function. T84 CFTR knockdown cells responded separately to lubiprostone and forskolin-IBMX in a methadone-sensitive and DASU-02-insensitive manner, indicating ClC-2 function. Low lubiprostone concentrations activated ClC-2, but not CFTR, and both channels were activated by forskolin-IBMX but have different inhibitor sensitivities. Methadone, but not DASU-02, inhibited ClC-2. DASU-02, but not methadone, inhibited CFTR. In T84 cells, both ClC-2 and CFTR are present and likely play roles in Cl(-) secretion.

A ClC-2G(2 alpha) Cl- channel was identified to be present in human lung and stomach, and a partial cDNA for this Cl- channel was cloned from a human fetal lung library. A full-length expressible human ClC-2G(2 alpha) cDNA was constructed by ligation of mutagenized expressible rabbit ClC-2G(2 alpha) cDNA with the human lung ClC-2G(2 alpha) cDNA, expressed in oocytes, and characterized at the single-channel level. Adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) treatment increased the probability of opening of the channel (Po). After PKA activation, the channel exhibited a linear (r = 0.99) current-voltage curve with a slope conductance of 22.1 +/- 0.8 pS in symmetric 800 mM tetraethylammonium chloride (TEACl; pH 7.4). Under fivefold gradient conditions of TEACl, a reversal potential of +21.5 +/- 2.8 mV was measured demonstrating anion-to-cation discrimination. As previously demonstrated for the rabbit ClC-2G(2 alpha) Cl- channel, the human analog, hClC-2G(2 alpha), was active at pH 7.4 as well as when the pH of the extracellular face of the channel (trans side of the bilayer; pHtrans) was asymmetrically reduced to pH 3.0. The extent of PKA activation was dependent on pHtrans. With PKA treatment, Po increased fourfold with a pHtrans of 7.4 and eightfold with a pHtrans of 3.0. Effects of sequential PKA addition followed by pHtrans reduction on the same channel suggested that the PKA- and pH-dependent increases in channel Po were separable and cumulative. Northern analysis showed ClC-2G(2 alpha) mRNA to be present in human adult and fetal lung and adult stomach, and quantitative reverse transcriptase-polymerase chain reaction showed this channel to be present in the adult human lung and stomach at about one-half the level found in fetal lung. The findings of the present study suggest that the ClC-2G(2 alpha) Cl- channel may play an important role in Cl- transport in the fetal and adult human lung.

The almost ubiquitously expressed ClC-2 chloride channel is activated by hyperpolarization and osmotic cell swelling. Osmotic swelling also activates a different class of outwardly rectifying chloride channels, and several reports point to a link between protein tyrosine phosphorylation and activation of these channels. This study examines the possibility that transforming growth factor-alpha (TGF-alpha) modulates ClC-2 activity in human colonic epithelial (T84) cells. TGF-alpha (0.17 nM) irreversibly inhibited ClC-2 current in nystatin-perforated whole cell patch-clamp experiments, whereas a superimposed reversible activation of the current was observed at 8.3 nM TGF-alpha. Both effects required activation of the intrinsic epidermal growth factor receptor (EGFR) tyrosine kinase activity, of phosphoinositide 3-kinase, and of protein kinase C. With microspectrofluorimetry of the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, TGF-alpha was shown to reversibly alkalinize T84 cells at 8.3 nM but not at 0.17 nM, suggesting that 8.3 nM TGF-alpha-induced alkalinization activates ClC-2 current. This study indicates that ClC-2 channels are targets for EGFR signaling in epithelial cells.

A novel volume-regulated hyperpolarization-activated chloride inward rectifier channel (Cl.ir) was identified in mammalian heart. To investigate whether ClC-2 is the gene encoding Cl.ir channels in heart, ClC-2 cDNAs cloned from rat (rClC-2) and guinea pig (gpClC-2) hearts were functionally characterized. When expressed in NIH/3T3 cells, full-length rClC-2 yielded inwardly rectifying whole-cell currents with very slow activation kinetics (time constants>> 1.7 s) upon hyperpolarization under hypotonic condition. The single-channel rClC-2 currents had a unitary slope conductance of 3.9 +/- 0.2 picosiemens. A novel variant with an in-frame deletion at the beginning of exon 15 that leads to a deletion of 45 bp (corresponding to 15 amino acids in alpha-helices O and P, rClC-2(Delta509-523)) was identified in rat heart. The relative transcriptional expression levels of full-length rClC-2 and rClC-2(Delta509-523) in rat heart were 0.018 +/- 0.003 and 0.028 +/- 0.006 arbitrary units, respectively, relative to glyceraldehyde-3-phosphate dehydrogenase (n = 5, p = nonsignificant). A similar partial exon 15 skipping with a deletion of 105 bp (35 amino acids in alpha-helices O-Q, gpClC-2(Delta509-543)) was also identified in guinea pig heart. Expression of both rClC-2(Delta509-523) and gpClC-2(Delta509-543) resulted in functional channels with phenotypic activation kinetics and many properties identical to those of endogenous Cl.ir channels in native rat and guinea pig cardiac myocytes, respectively. Intracellular dialysis of anti-ClC-2 antibody inhibited expressed ClC-2 channels and endogenous Cl.ir currents in native rat and guinea pig cardiac myocytes. These results demonstrate that novel deletion variants of ClC-2 due to partial exon 15 skipping may be expressed normally in heart and contribute to the formation of endogenous Cl.ir channels in native cardiac cells.

As the molecular revolution continues to inform a deeper understanding of disease mechanisms and pathways, there exist unprecedented opportunities for translating discoveries at the bench into novel therapies for improving human health. Despite the availability of several different classes of antihypertensive medications, only about half of the 67 million Americans with hypertension manage their blood pressure appropriately. A broader selection of structurally diverse antihypertensive drugs acting through different mechanisms would provide clinicians with greater flexibility in developing effective treatment regimens for an increasingly diverse and aging patient population. An emerging body of physiological, genetic, and pharmacological evidence has implicated several renal ion-transport proteins, or regulators thereof, as novel, yet clinically unexploited, diuretic targets. These include the renal outer medullary potassium channel, ROMK (Kir1.1), Kir4.1/5.1 potassium channels, ClC-Ka/b chloride channels, UTA/B urea transporters, the chloride/bicarbonate exchanger pendrin, and the STE20/SPS1-related proline/alanine-rich kinase (SPAK). The molecular pharmacology of these putative targets is poorly developed or lacking altogether; however, recent efforts by a few academic and pharmaceutical laboratories have begun to lessen this critical barrier. Here, we review the evidence in support of the aforementioned proteins as novel diuretic targets and highlight examples where progress toward developing small-molecule pharmacology has been made.

Little is known about how genetic variation at the nucleotide level contributes to competitive fitness within species. During a 6,000-generation study of Bacillus subtilis evolved under relaxed selection for sporulation, a new strain, designated WN716, emerged with significantly different colony and cell morphologies; loss of sporulation, competence, acetoin production, and motility; multiple auxotrophies; and increased competitive fitness (H. Maughan and W. L. Nicholson, Appl. Environ. Microbiol. 77:4105-4118, 2011). The genome of WN716 was analyzed by OpGen optical mapping, whole-genome 454 pyrosequencing, and the CLC Genomics Workbench. No large chromosomal rearrangements were found; however, 34 single-nucleotide polymorphisms (SNPs) and +1 frameshifts were identified in WN716 that resulted in amino acid changes in coding sequences of annotated genes, and 11 SNPs were located in intergenic regions. Several classes of genes were affected, including biosynthetic pathways, sporulation, competence, and DNA repair. In several cases, attempts were made to link observed phenotypes of WN716 with the discovered mutations, with various degrees of success. For example, a +1 frameshift was identified at codon 13 of sigW, the product of which (SigW) controls a regulon of genes involved in resistance to bacteriocins and membrane-damaging antibiotics. Consistent with this finding, WN716 exhibited sensitivity to fosfomycin and to a bacteriocin produced by B. subtilis subsp. spizizenii and exhibited downregulation of SigW-dependent genes on a transcriptional microarray, consistent with WN716 carrying a knockout of sigW. The results suggest that propagation of B. subtilis for less than 2,000 generations in a nutrient-rich environment where sporulation is suppressed led to rapid initiation of genomic erosion.

BACKGROUND

The aim of this study was to compare micropuncture laparoscopic cholecystectomy (MPLC), with three 3.3-mm cannulas and one 10-mm cannula with conventional laparoscopic cholecystectomy (CLC).

METHODS

Patients were randomized to undergo either CLC or MPLC. The duration of each operative stage and the procedure were recorded. Interleukin-6 (IL-6), adrenocorticotropic hormone (ACTH), and vasopressin were sampled for 24 h. Visual analogue pain scores (VAPS) and analgesic consumption were recorded for 1 week. Pulmonary function and quality of life (EQ-5D) were monitored for 4 weeks. Statistical analysis was performed using the Mann-Whitney test or Fisher's exact test. Results are expressed as median (interquartile range).

RESULTS

Forty-four patients entered the study, but four were excluded due to unsuspected choledocholithiasis (n = 3) or the need to reschedule surgery (n = 1). The groups were comparable in terms of age, duration of symptoms, and indications for surgery. Total operative time was similar (CLC, 63 [52-81] min vs MPLC 74 [58-95] min; p = 0.126). However, time to place the cannulas after skin incision (CLC, 5:42 [3:45-6:37] min vs MPLC, 7:38 [5:57-10:15] min; p = 0.015) and to clip the cystic duct after cholangiography (CLC, 1:05 [0:40-1:35] min vs MPLC, 3:45 [2:26-7:49] min; p <0.001) were significantly longer for MPLC. Six CLC patients and one MPLC patient required postoperative parenteral opiates (p = 0.04). Oral analgesic consumption was similar in both groups (p = 0.217). Median VAPS were lower at all time points for MPLC, but this finding was not significant (p = 0.431). There were no significant differences in postoperative stay, IL-6, ACTH or vasopressin responses, pulmonary function, or EQ-5D scores.

CONCLUSIONS

The thinner instruments did not significantly increase the total duration of the procedure. MPLC reduced the use of parenteral analgesia postoperatively, which may prove beneficial for day case patients, but it did not have a significant impact on laboratory variables, lung function or quality of life.

Chloride channels play important roles in vital cellular signalling processes contributing to homeostasis in both excitable and non-excitable cells. Since 1987, more than ten ion channel genes have been identified as causing human hereditary diseases among them the genes for the voltage-dependent chloride channel ClC-1 (myotonia) and the cystic fibrosis transmembrane conductance regulator (CFTR) protein (cystic fibrosis). The CFTR gene was cloned in 1989 and its protein product identified as an ATP-gated and phosphorylation-regulated chloride channel during the following two years. Since then, searching for potent and specific small molecules able to modulate normal and mutated CFTR has become a crucial endpoint in the field for both our understanding of the physiological role that CFTR plays in epithelial cells and more importantly for the development of therapeutic agents to cure cystic fibrosis (CF). It is predicted that a pharmacological approach would help not only to restore the defective transport activity of mutant CFTR but also to correct the regulatory function of CFTR. This review describes the evolution of CFTR pharmacology and how during the last five years, high throughput screening assays have been developed to identify novel molecules, some of them probably constituting a reservoir of future therapeutic agents for CF.

The kidney plays an important role in osmoregulation in freshwater teleosts, which are exposed to the danger of osmotic loss of Na(+) and Cl(-). However, ion-transport mechanisms in the kidney are poorly understood, and ion transporters of the fish nephron have not been identified thus far. From Mozambique tilapia, Oreochromis mossambicus, we have cloned a chloride channel, which is a homologue of the mammalian kidney-specific chloride channel, ClC-K. The cDNA of the channel, named OmClC-K, encodes a protein whose amino acid sequence has high homology to Xenopus and mammalian ClC-K (Xenopus ClC-K, 41.8%; rat ClC-K2, 40.9%; rat ClC-K1, 40.1%). The mRNA of OmClC-K was expressed exclusively in the kidney, and the expression level of mRNA was increased more in freshwater-adapted fish than seawater-adapted fish. The immunohistochemical study using a specific antibody showed that OmClC-K-positive cells were specifically located in the distal nephron segments. Immunoelectron microscopy further showed that immunoreaction of OmClC-K was recognizable on the structure of basolateral membrane infoldings in the distal tubule cells. The localization of OmClC-K and its induction in hypoosmotic media suggest that OmClC-K is involved in Cl(-) reabsorption in the distal tubule of freshwater-adapted tilapia.

This review summarizes the current knowledge of Cl(-) transport in vascular smooth muscle cells (VSMCs). VSMCs accumulate Cl(-) intracellularly using two secondary-active transport mechanisms. The Cl(-) equilibrium potential is more positive than the resting membrane potential enabling Cl(-) to be a depolarizing ion upon activation of a Cl(-) conductance. Cl(-) currents are involved in different vascular responses suggesting a number of different Cl(-) channels. All known Cl(-) channel families, with the exception of the GABA-/glycine-receptor family, have been identified in VSMCs. At least one member of the voltage-activated ClC family - ClC-3 - has been suggested to be involved in myogenic constriction, in cell proliferation and to have an anti-apoptotic action. The cystic fibrosis transmembrane conductance regulator is also demonstrated in VSMCs. The molecular identity of the major anion conductance in VSMCs - a Ca(2+)-activated Cl(-) current - is uncertain. Several candidates have been suggested with bestrophin and TMEM16 protein families the current favorites. Specific pharmacological tools are lacking for Cl(-) channels but recent molecular biology developments have made selective gene manipulations possible. A continuing quest within the vascular research field is to explicitly demonstrate the coupling between a putative channel protein and an endogenous Cl(-) current and the importance of these for specific functions.

Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.