BACKGROUND

SNP (Single Nucleotide Polymorphism) markers are rapidly becoming the markers of choice for applications in breeding because of next generation sequencing technology developments. For SNP development by NGS technologies, correct assembly of the huge amounts of sequence data generated is essential. Little is known about assembler's performance, especially when dealing with highly heterogeneous species that show a high genome complexity and what the possible consequences are of differences in assemblies on SNP retrieval. This study tested two assemblers (CAP3 and CLC) on 454 data from four lily genotypes and compared results with respect to SNP retrieval.

RESULTS

CAP3 assembly resulted in higher numbers of contigs, lower numbers of reads per contig, and shorter average read lengths compared to CLC. Blast comparisons showed that CAP3 contigs were highly redundant. Contrastingly, CLC in rare cases combined paralogs in one contig. Redundant and chimeric contigs may lead to erroneous SNPs. Filtering for redundancy can be done by blasting selected SNP markers to the contigs and discarding all the SNP markers that show more than one blast hit. Results on chimeric contigs showed that only four out of 2,421 SNP markers were selected from chimeric contigs.

CONCLUSIONS

In practice, CLC performs better in assembling highly heterogeneous genome sequences compared to CAP3, and consequently SNP retrieval is more efficient. Additionally a simple flow scheme is suggested for SNP marker retrieval that can be valid for all non-model species.

Capillary liquid chromatography-mass spectrometry (cLC-MS) was coupled on-line to microdialysis sampling to monitor endogenous acetylcholine (ACh) from the rodent brain. In vivo microdialysate sampled at 0.6 microL/min from the striatum of ketamine or chloral hydrate anesthetized rats was loaded onto a sample loop and then injected onto a approximately 5 cm long strong cation exchange (SCX) capillary column. A step gradient was used to separate the analyte from ionization suppressing salts contained in dialysate in 2.4 min. Sampling coupled on-line with cLC-MS allowed for high temporal resolution (data points at 2.4 min intervals), good reproducibility (10-15% relative standard deviation, R.S.D.), and sensitive limits of detection (0.04 nM or 8 amol injected). The method successfully monitored basal and stimulated levels (induced by increased K+ concentrations) of ACh from the anesthetized rat without necessitating perfusion of an acetylcholinesterase (AChE) inhibitor. Absolute and percent basal levels of ACh from rats receiving different anesthetics were also compared.

Mutations in the CLCN5 gene have been demonstrated in three disorders of hypercalciuric nephrolithiasis, i.e., Dent's disease, X-linked recessive nephrolithiasis, and X-linked recessive hypophosphatemic rickets. Recently, a number of Japanese children with low molecular weight proteinuria (LMWP) showing symptoms similar to those shown by patients with Dent's disease in British families have also been reported to have mutations in the CLCN5 gene. The present study examines five unrelated Japanese families with LMWP, two of which lacked any signs other than LMWP, and three of which had several signs other than LMWP, i.e., hypercalciuria, aminoaciduria, hypophosphatemia, and rickets. One nonsense (E118X) and one missense (W22G) mutation were found in three patients in the two families having only LMWP. One genomic deletion including exons 5 to 8 in the CLCN5 gene was found in a patient with hypophosphatemic rickets, and a nonsense mutation (R347X) was found in one patient with LMWP and slight hypercalciuria. No mutations of the exons and exon-intron boundaries in the CLCN5 gene were found in one patient with LMWP, aminoaciduria, and hypokalemia. In addition to the predicted loss of chloride channel function in these nonsense and deletion mutations, the loss of function in the missense mutation W22G was confirmed in the Xenopus oocyte expression system. These results clarified four novel mutations in the CLCN5 genes, and additionally suggested that the loss-of-function mutation of the CLCN5 does not necessarily lead to hypercalciuria and nephrocalcinosis in the early stage of the disease, and that LMWP is an early and essential manifestation of disorders of the CLC-5 chloride channel.

BACKGROUND

Dent's disease is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, rickets and eventual renal failure. The disease is caused by mutations in the X-linked chloride channel CLCN5 gene, which encodes a 746-amino-acid protein expressed in renal tubules. These mutations have been reported in unrelated families from the UK, USA, Japan and other countries. We were interested in identifying additional mutations in the CLCN5 coding region of Spanish patients with Dent's disease.

METHODS

Five patients from three unrelated Spanish families were studied. Leukocyte genomic DNA from patients and their relatives was used with CLCN5-specific primers for polymerase chain reaction amplification of the coding region and exon-intron boundaries. Amplified products were analysed by single-strand conformational polymorphism analysis, DNA sequencing and restriction enzyme analysis.

RESULTS

Low-molecular-weight proteinuria and hypercalciuria were detected in all the patients, nephrocalcinosis in two patients, and rickets or osteopenia in three patients. We identified three new CLCN5 mutations consisting of two nonsense mutations, Leu433Stop and Arg718Stop, and an insertional frameshift mutation, 65insT, which results in a stop at codon 98. These three mutations predict truncated ClC-5 proteins that, respectively, lack 314, 649 and 28 amino acids at the carboxy terminus, and are likely to result in loss of function. These mutations were shown to co-segregate with the disease in each of the three families.

CONCLUSIONS

Our study is the first to characterize mutations in the CLCN5 gene in Spanish patients with Dent's disease and expands the spectrum of CLCN5 mutations associated with this disease.

Circulating schistosome antigens (CSA) and circulating immune complexes (CLC) were investigated in rats infected with Schistosoma mansoni. The radioimmunoprecipitation-polyethylene glycol (PEG) assay (RIPEGA), with 125I-labelled anti-S. mansoni anti-serum, detected CSA during two distinct periods of the infection; the first between the 11th and the 14th week of infection and the second between the 11th and 14th week after infection. The CH50 deviation test revealed the presence of CIC in sera from infected rats, approximately at the two periods when CSA were detected. At 6 weeks of infection, the levels of CIC in infected rats were not different from those in control rats. However, a more sensitive method characterized IgG2a in C1q-binding C1C from infected rats. At weeks 5 and 6, IgE immune complexes were also detected in the serum from infected rats. In fact, the use of RIPEGA on the material eluted from infected rat serum after passage through an anti-IgE immunosorbent showed the presence of schistosome antigen at week 4, and at higher levels at week 6. Levels of 50% haemolytic complement in infected rat serum were lowered between the 2nd and the 4th week, the 5th and the 8th week and after the 12th week of infection. The possible role played by CIC in the protective mechanisms to a S. mansoni challenge infection in rats is discussed.

Hepatocytes possess chloride channels at the plasma membrane and in multiple intracellular compartments. These channels are required for cell volume regulation and acidification of intracellular organelles. Evidence also supports a role of chloride channels in modulation of apoptosis and cell growth. Swelling- and Ca(2+)-activated chloride channels have been identified in hepatocyte plasma membranes, and chloride channels have been observed in the membranes of lysosomes, endosomes, Golgi, endoplasmic reticulum, mitochondria, and the nucleus. This review summarizes the functions of these channels and discusses the specific channel molecules they may represent. Chloride channel molecules shown to be expressed in hepatocytes include members of the ClC channel family (ClC-2, ClC-3, ClC-5, and ClC-7), members of the newly identified CLIC family of intracellular chloride channels (CLIC-1 and CLIC-4), the mitochondrial voltage-dependent anion channel, and a newly identified intracellular channel, MCLC (Mid-1 related chloride channel). Current understanding does not include a molecular identification of most of the observed channel functions, but details of the molecular properties of these channel molecules should allow future identification and further understanding of chloride channel function in hepatocytes.

In mature vertebrate muscle, the chloride channel Clc-1 is necessary for the stabilization of the resting potential. Its functional defect leads to the disease myotonia. The ADR mouse (phenotype ADR, genotype adr/adr) is an animal model for human myotonias. The adr gene is a member of a family of non-complementing recessive autosomal mutations ("alleles" of adr) that cause myotonia in the mouse. The standard allele adr has arisen by the insertion of a retroposon into the chloride channel gene Clc-1 (Steinmeyer, K., Klocke, R., Ortland, C., Gronemeier, M., Jockusch, H., Gründer, S., and Jentsch, T. J. (1991) Nature 354, 304-308). In order to study the nature of two other alleles, adrmto and adrK, we have analyzed overlapping Clc-1 cDNA amplification products by the hydroxylamine and osmium tetroxide modification technique and direct sequencing. A comparison between ADR*MTO and C57BL/6 wild type showed six base pair substitutions, one of which resulted in a stop codon in position 47, whereas the five others are either silent or lead to amino acid substitutions in non-conserved regions of the Clc-1 sequence and were already present in the wild type inbred SWR/J strain from which adrmto was derived. The detection of the stop codon in the adrmto allele is further indication of the identity of the Clc-1 chloride channel with the adr myotonia gene in the mouse, because a chain termination close to the N terminus would necessarily destroy gene function. For the ethylnitrosourea-induced mutation adrK, an Ile->>Thr exchange in codon 553 was identified. As this affects a conserved residue within a highly conserved region of the Clc-1 gene, a functional significance of this residue is suggested.

The whole-cell patch-clamp technique was used to study the effect of protein kinase C (PKC) stimulation and alpha-adrenergic agonists on the swelling-induced chloride current (ICl,swell) in canine atrial cells. ICl,swell was activated by positive-pressure inflation. 4beta-Phorbol 12, 13-dibutyrate (PDBu) concentration-dependently stimulated ICl,swell. PDBu (500 nM) increased the current density of ICl,swell from 9.1+/-1.3 to 24.2+/-4.8 pA/pF at +20 mV (n=4). This effect developed slowly, reaching a steady-state after more than 5 min of exposure. 4alpha-Phorbol 12, 13-dibutyrate (4alpha-PDBu, 500 nM), an inactive analogue of PDBu, did not affect ICl,swell. The effect of PDBu was inhibited by bisindolylmaleimide I. After down regulation of PKC by phorbol 12-myristate 13-acetate (PMA, 1.6 microM, 24 h), ICl,swell no longer responded to PDBu (n=4). Neither the basal whole-cell current (prior to cell inflation) nor inflation-induced ICl,swell were affected by PKC down regulation. Phenylephrine did not affect ICl,swell. We conclude that PKC activity stimulates and does not prevent the activation of dog atrial ICl,swell. These results contrast with reports of PKC-dependent inhibition of rabbit atrial ICl,swell and currents conducted by ClC-3, a putative clone for ICl,swell. The data suggest species-dependent variations in the modulation of cardiac ICl,swell by PKC.

Myotonia congenita is a hereditary muscle disorder caused by mutations in the human voltage-gated chloride (Cl(-)) channel CLC-1. Myotonia congenita can be inherited in an autosomal recessive (Becker type) or dominant (Thomsen type) fashion. One hypothesis for myotonia congenita is that the inheritance pattern of the disease is determined by the functional consequence of the mutation on the gating of CLC-1 channels. Several disease-related mutations, however, have been shown to yield functional CLC-1 channels with no detectable gating defects. In this study, we have functionally and biochemically characterized a myotonia mutant: A531V. Despite a gating property similar to that of wild-type (WT) channels, the mutant CLC-1 channel displayed a diminished whole-cell current density and a reduction in the total protein expression level. Our biochemical analyses further demonstrated that the reduced expression of A531V can be largely attributed to an enhanced proteasomal degradation as well as a defect in protein trafficking to surface membranes. Moreover, the A531V mutant protein also appeared to be associated with excessive endosomal-lysosomal degradation. Neither the reduced protein expression nor the diminished current density was rescued by incubating A531V-expressing cells at 27°C. These results demonstrate that the molecular pathophysiology of A531V does not involve anomalous channel gating, but rather a disruption of the balance between the synthesis and degradation of the CLC-1 channel protein.

OBJECTIVE

ClC-K kidney Cl(-) channels are important for renal and inner ear transepithelial Cl(-) transport, and are potentially interesting pharmacological targets. They are modulated by niflumic acid (NFA), a non-steroidal anti-inflammatory drug, in a biphasic way: NFA activates ClC-Ka at low concentrations, but blocks the channel above approximately 1 mM. We attempted to identify the amino acids involved in the activation of ClC-Ka by NFA.

METHODS

We used site-directed mutagenesis and two-electrode voltage clamp analysis of wild-type and mutant channels expressed in Xenopus oocytes. Guided by the crystal structure of a bacterial CLC homolog, we screened 97 ClC-Ka mutations for alterations of NFA effects.

RESULTS

Mutations of five residues significantly reduced the potentiating effect of NFA. Two of these (G167A and F213A) drastically altered general gating properties and are unlikely to be involved in NFA binding. The three remaining mutants (L155A, G345S and A349E) severely impaired or abolished NFA potentiation.

CONCLUSIONS

The three key residues identified (L155, G345, A349) are localized in two different protein regions that, based on the crystal structure of bacterial CLC homologs, are expected to be exposed to the extracellular side of the channel, relatively close to each other, and are thus good candidates for being part of the potentiating NFA binding site. Alternatively, the protein region identified mediates conformational changes following NFA binding. Our results are an important step towards the development of ClC-Ka activators for treating Bartter syndrome types III and IV with residual channel activity.

GlialCAM, a glial cell adhesion molecule mutated in megalencephalic leukoencephalopathy with subcortical cysts, targets the CLC-2 Cl(-) channel to cell contacts in glia and activates CLC-2 currents in vitro and in vivo. We found that GlialCAM clusters all CLC channels at cell contacts in vitro and thus studied GlialCAM interaction with CLC channels to investigate the mechanism of functional activation. GlialCAM slowed deactivation kinetics of CLC-Ka/barttin channels and increased CLC-0 currents opening the common gate and slowing its deactivation. No functional effect was seen for common gate deficient CLC-0 mutants. Similarly, GlialCAM targets the common gate deficient CLC-2 mutant E211V/H816A to cell contacts, without altering its function. Thus, GlialCAM is able to interact with all CLC channels tested, targeting them to cell junctions and activating them by stabilizing the open configuration of the common gate. These results are important to better understand the physiological role of GlialCAM/CLC-2 interaction.

Gill mitochondrion-rich (MR) cells contain different molecules to carry out functionally distinct mechanisms. To date, the putative mechanism of Cl(-) uptake through the basolateral chloride channel, however, is less understood. To clarify the Cl(-)-absorbing mechanism, this study explored the molecular and morphological alterations in branchial MR cells of tilapia acclimated to seawater (SW), freshwater (FW), and deionized water (DW). Scanning electron microscopic observations revealed that three subtypes of MR cells were exhibited in gill filament epithelia of tilapia. Furthermore, in DW-acclimated tilapia, the subtype I (ion-absorbing subtype) of MR cells predominantly occurred in gill filament as well as lamellar epithelia. Whole-mount double immunofluorescent staining revealed that branchial ClC-3-like protein and Na(+)/K(+)-ATPase (NKA), the basolateral marker of MR cells, were colocalized in tilapia. In SW-acclimated tilapia, all MR cells of gill filament epithelia exhibited faint fluorescence of ClC-3-like protein. In contrast, only some MR cells in gill filament epithelia of FW and DW tilapia expressed basolateral ClC-3-like protein; however, the fluorescence was more intense in FW and DW tilapia than in SW fish. In hyposmotic groups, the number of MR cells immunopositive for ClC-3-like protein was significantly higher in DW-exposed tilapia. Meanwhile, in gill lamellar epithelia of DW tilapia, all MR cells (subtype I) were ClC-3-like protein immunopositive. Double immunostaining of ClC-3-like protein and Na(+)/Cl(-) cotransporter (NCC) revealed that basolateral ClC-3-like protein and apical NCC were colocalized in some MR cells in FW and DW tilapia. Moreover, both mRNA and protein amounts of branchial ClC-3-like protein were significantly higher in DW-acclimated tilapia. To identify whether the expression of branchial ClC-3-like protein responded to changes in environmental [Cl(-)], tilapia were acclimated to artificial waters with normal [Na(+)]/[Cl(-)] (control), lower [Na(+)] (low Na), or lower [Cl(-)] (low Cl). Immunoblotting of crude membrane fractions for gill ClC-3-like protein showed that the protein abundance was evidently enhanced in tilapia acclimated to the low-Cl environment compared with the other groups. Our findings integrated morphological and functional classifications of ion-absorbing MR cells and indicated that ion-deficient water elevated the numbers of subtype I MR cells in both filament and lamellar epithelia of gills with positive ClC-3-like protein immunostaining and increased the expression levels of ClC-3-like protein. This study is the first to illustrate the exhibition of a basolateral chloride channel potentially responsible for Cl(-) absorption in the ion-absorbing subtype of gill MR cells of tilapia.

ClC-4 gene was isolated as a putative Cl(-) channel. Due to a lack of functional expression of ClC-4, its physiological role remains unknown. We isolated a human ClC-4 clone (hClC-4sk) from human skeletal muscles and stably transfected it to Chinese hamster ovary cells. Whole cell patch-clamp studies showed that the hClC-4sk channel was activated by external acidic pH and inhibited by DIDS. It passed a strong outward Cl(-) current with a permeability sequence of I(-)>> Cl(-)>> F(-). The hClC-4sk has consensus sites for phosphorylation by protein kinase A (PKA); however, stimulation of PKA had no effect on the currents. hClC-4sk mRNA was expressed in excitable tissues, such as heart, brain, and skeletal muscle. These functional characteristics of hClC-4sk provide a clue to its physiological role in excitable cells.

BACKGROUND

Natural orifice transluminal endoscopic surgery (NOTES) has the potential to reduce postoperative pain. We compared postoperative pain in the hybrid NOTES procedure transvaginal video-assisted cholecystectomy (TVC) with standard conventional laparoscopic cholecystectomy (CLC).

METHODS

Single-center, double-blind, randomized controlled trial in a level II hospital between June 2008 and June 2012.

METHODS

Female patients, older than 18 years of age with symptomatic cholecystolithiasis were randomized to receive either TVC or CLC. The follow-up period was 7 days and the primary outcome of the study was postoperative pain. We hypothesized that there is no reduction of pain (Visual Rating Scale ≥1) while resting or coughing over a 48-h period after the operation. Secondary outcome included wound infections, complications, and patient reported outcomes. Sealed envelopes with computer-generated randomization information were kept for allocation in theater. All patients received opaque wound dressing, as in standard four-trocar cholecystectomy and a vaginal tamponade. Theater protocol and surgical notes were kept separate after the procedure.

RESULTS

Overall, 97 of 426 patients assessed for participation were randomized for either TVC or CLC. A total of 41 patients had a TVC and 51 had a CLC. Five patients were excluded from the analysis. There was no difference in age, body mass index, American Society of Anesthesiologists (ASA) grade, or hospital stay, but anesthetic and surgical times were significantly longer in TVC (p < 0.001). There was no statistical difference in postoperative pain between the two groups while resting or coughing. Complications included conversion to laparotomy, bleeding, wound infections, and re-admission. No difference in the rate of complications between the two groups was seen. Overall, 86 and 93% of CLC and TVC patients, respectively, would recommend the procedure to other patients.

CONCLUSIONS

In this study, no significant difference in pain on days 1 and 2 postoperatively between the two methods was found. The safety profile of TVC is comparable to CLC, and TVC patients would generally recommend this procedure to other patients.

OBJECTIVE

To explore women's preferences for, and trade-offs between, key attributes of intrapartum care models.

METHODS

Mixed-methods study using discrete choice experiments (DCEs) and focus groups.

METHODS

The North of Scotland.

METHODS

Women from the catchment areas of eight rural maternity units in the North of Scotland.

METHODS

Based on current policy, 'model of care' and 'time travelled' were selected as key attributes of intrapartum care in remote and rural settings. A DCE questionnaire explored women's preferences for and trade-offs between these attributes. Focus groups validated the DCE attributes and provided valuable information about the drivers of women's preferences for place of delivery.

METHODS

Preferences for attributes of intrapartum care.

RESULTS

Eight focus groups were conducted, and 877 eligible women completed the questionnaire. Overall, the DCE results found women preferred delivery in a unit to home birth and consultant-led care (CLC) to midwife-managed care (MMC). Women preferring CLC associated it with covering every eventuality and increased safety. Although women preferred shorter travel times, trade-offs indicated a willingness to travel for approximately 2 hours to get one's preferred choice. Focus group findings and subgroup DCE analysis showed heterogeneity of preferences related to experience, risk status, geographic location, perception of care and family circumstances.

CONCLUSIONS

In contrast to service redesign offering local midwife-managed intrapartum care, most rural women in our study expressed a preference to give birth in hospital and have CLC because they felt safer. Women were willing to travel for this but within limits. Qualitative results showed that women's preferences were influenced by their home and family context, beliefs and previous pregnancy experiences. Challenges for service redesign are to provide comprehensive obstetric services within acceptable travel time, while responding to the heterogeneity of women's preferences.

Long standing confusion exists in the terminology of hypokalaemic salt-losing tubulopathies (SLTs). SLTs are autosomal recessively transmitted and characterized by normotensive secondary hyperreninism/hyperaldosteronism with hypokalaemic metabolic alkalosis. Historically, four phenotypical variants have been described: (1) the (classic) Bartter syndrome (cBS), (2) the hypomagnesaemic hypocalciuric Gitelman syndrome (GS), (3) the hypercalciuric hyperprostaglandin-E-syndrome (HPS) or antenatal Bartter syndrome (aBS) and (4) the hyperprostaglandin-E-syndrome with sensorineural deafness (HPS + SND). The latter two syndromes are the most severe variants with antenatal manifestation with polyhydramnios and life-threatening course of salt- and water-loss. Defects in five renal membrane proteins involved in electrolyte reabsorption have been identified: In HPS-patients mutations in (1) either the furosemide-sensitive sodium-potassium-chloride cotransporter NKCC2, or (2) in the potassium channel ROMK have been identified, and (3) HPS + SND is caused by mutations in the beta-subunit of the chloride channels ClC-Kb and -Ka (named barttin), all mimicking the major pharmacological effects of furosemide with minor potassium-wasting in ROMK-patients as seen in patients treated with simultaneous furosemide and amiloride, and minor calcium-wasting in Barttin-patients resembling the combination of furosemide and thiazides. (4) cBS is caused by mutations in the chloride channel ClC-Kb with similar clinical characteristics as seen under combination of thiazides and furosemide, (5) GS is caused by mutations in the thiazide-sensitive sodium-chloride cotransporter NCCT resembling the effect of long-term thiazide administration.

CONCLUSIONS

The combination of pharmacology and genetics suggests a new terminology for the above described SLTs: Furosemide-like-SLT for HPS caused by NKCC2-mutations, furosemide/amiloride-like-SLT for HPS caused by ROMK-mutations, furosemide/thiazide-like-SLT for HPS + SND, thiazide/furosemide-like-SLT for cBS, and thiazide-like-SLT for GS.

ATP binding enhances the activity of ClC-5, the transporter mutated in Dent disease, a disease affecting the renal proximal tubule. Previously, the ATP binding site was revealed in x-ray crystal structures of the cytoplasmic region of this membrane protein. Disruption of this site by mutagenesis (Y617A-ClC-5) reduced the functional expression and ATP-dependent regulation of the full-length transporter in Xenopus oocytes. However, insight into the conformational changes underlying ATP-dependent regulation is lacking. Here, we show that ATP binding induces a change in protein conformation. Specifically, small angle x-ray scattering experiments indicate that ATP binding promotes a clamp-like closure of the isolated ClC-5 carboxyl-terminal region. Limited proteolysis studies show that ATP binding induces conformational compaction of the carboxyl-terminal region in the intact membrane protein as well. In the context of fibroblasts and proximal tubule epithelial cells, disruption of the ATP binding site in full-length ClC-5 (Y617A-ClC-5) led to a defect in processing and trafficking out of the endoplasmic reticulum. These latter findings account for the decrease in functional expression previously reported for this ATP-binding mutant and prompt future study of a model whereby conformational compaction caused by ATP binding promotes biosynthetic maturation.

Chloride channels belonging to the ClC family are ubiquitous and participate in a wide variety of physiological and pathophysiological processes. To define sequence segments in ClC channels that contribute to the formation of their ion conduction pathway, we employed a combination of site-directed mutagenesis, heterologous expression, patch clamp recordings, and chemical modification of the human muscle ClC isoform, hClC-1. We demonstrate that a highly conserved 8-amino acid motif (P3) located in the linker between transmembrane domains D2 and D3 contributes to the formation of a wide pore vestibule facing the cell interior. Similar to a previously defined pore region (P1 region), this segment functionally interacts with the corresponding segment of the contralateral subunit. The use of cysteine-specific reagents of different size revealed marked differences in the diameter of pore-forming regions implying that ClC channels exhibit a pore architecture quite similar to that of certain cation channels, in which a narrow constriction containing major structural determinants of ion selectivity is neighbored by wide vestibules on both sides of the membrane.

The ClC family of Cl(-) channels and transporters comprises membrane proteins ubiquitously present in species ranging from prokaryotes to mammals. The recently solved structures of the bacterial ClC proteins have provided a good model to guide the functional experiments for the eukaryotic Cl(-) channels. Theoretical calculations based on the bacterial ClC structures have identified several residues critical for the Cl(-) binding energy in the Cl(-) transport pathway. It was speculated that the corresponding residues in eukaryotic Cl(-) channels might play similar roles for the channel functions. In this study, we made a series of mutations in three such residues in eukaryotic ClC Cl(-) channels (K149, G352, and H401 in ClC-0) and studied the functional consequences on the channel properties. A cysteine modification approach was also employed to evaluate the electrostatic effects of the charge placed at these three positions. The experimental results revealed that among the three residues tested, K149 plays the most important role in controlling both the gating and the permeation functions of ClC-0. On the other hand, mutations of H401 alter the channel conductance but not the gating properties, while mutations of G352 result in very little functional consequence. The mutation of K149 into a neutral residue leucine (K149L) shifts the activation curve and leads to flickery channel openings. The anion permeability ratios derived from bi-ionic experiments are also significantly altered in that the selectivity of Cl(-) over other anions is decreased. Furthermore, removing the positive charge at this position reduces and increases, respectively, the accessibility of the negatively and positively charged methane thiosulfonate reagents to the pore. The control of the accessibility to charged MTS reagents and the regulation of the anion permeation support the idea that K149 exerts an electrostatic effect on the channel function, confirming the prediction from computational studies.

The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent K(D) of approximately 20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function.

Retinal pigment epithelium (RPE) possesses regulated chloride channels that are crucial for transepithelial fluid and ion transport. At present, little is known about the molecular nature of chloride channels in human adult RPE (haRPE) or the effects of oxidative stress on membrane conductance properties. In the present study, we assessed ClC channel and cystic fibrosis transmembrane conductance regulator (CFTR) expression and membrane chloride conductance properties in haRPE cells. ClC-5, ClC-3, ClC-2, and CFTR mRNA expression was confirmed with RT-PCR analysis, and protein expression was detected with Western blot analysis and immunofluorescence microscopy. Whole cell recordings of primary cultures of haRPE showed an outwardly rectifying chloride current that was inhibited by the oxidant H(2)O(2). The inhibitory effects of H(2)O(2) were reduced in cultured human RPE cells that were incubated with precursors of glutathione synthesis or that were stably transfected to overexpress glutathione S-transferase. These findings indicate a possible role for ClC channels in haRPE cells and suggest possible redox modulation of human RPE chloride conductances.

We report the application of nanoelectrospray ionization tandem mass spectrometry (nES-MS/MS) and capillary LC/microelectrospray MS/MS (cLC/&mgr;ES-MS/MS) for sequencing sulfonic acid derivatized tryptic peptides. These derivatives were specifically prepared to facilitate low-energy charge-site-initiated fragmentation of C-terminal arginine-containing peptides, and to enhance the selective detection of a single series of y-type fragment ions. Both singly and doubly protonated peptides were analyzed by MS/MS and the results were compared with those from their derivatized counterparts. Model peptides and peptides from tryptic digests of gel-isolated proteins were analyzed. Derivatized singly protonated peptides fragment in the same way by nES-MS/MS as they do by post-source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD-MALDI-MS). They produce fragment ion spectra dominated by y-ions, and the simplified spectra are readily interpreted de novo. Doubly protonated peptides fragment in much the same way as their non-derivatized doubly protonated counterparts. The fragmentation of doubly protonated derivatives is especially useful for sequencing peptides that possess a proline residue near the N-terminus of the molecule. The singly protonated forms of these proline-containing derivatives often show enhanced fragmentation on the N-terminal side of the proline and considerably reduced fragmentation on the C-terminal side. In addition, sulfonic acid derivatization increases the in-source fragmentation of arginine-containing peptides. This could be useful for sequence verification and sequence tagging for use in single stage mass spectrometry. Copyright 2000 John Wiley & Sons, Ltd.

Chloride channel protein (ClC)-6a and ClC-6c, a kidney-specific splice variant with a truncated C-terminus, are proteins that belong structurally to the family of voltage-dependent chloride channels. Attempts to characterize functionally ClC-6a or ClC-6c in Xenopus oocytes have so far been negative. Similarly, expression of both ClC-6 isoforms in mammalian cells failed to provide functional information. One possible explanation of these negative results is that ClC-6 is an intracellular chloride channel rather than being located in the plasma membrane. We therefore studied the subcellular location of ClC-6 isoforms by transiently transfecting COS and CHO cells with epitope-tagged versions of ClC-6a and ClC-6c. Confocal imaging of transfected cells revealed for both ClC-6 isoforms an intracellular distribution pattern that clearly differed from the peripheral location of CD2, a plasma-membrane glycoprotein. Furthermore, dual-labelling experiments of COS cells co-transfected with ClC-6a or -6c and the sarco/endoplasmic-reticulum Ca2+ pump (SERCA2b) indicated that the ClC-6 isoforms co-localized with the SERCA2b Ca2+ pump. Thus ClC-6a and ClC-6c are intracellular membrane proteins, most likely residing in the endoplasmic reticulum. In view of their structural similarity to proven chloride channels, ClC-6 isoforms are molecular candidates for intracellular chloride channels.

Mutations in ClC-5 (chloride channel 5), a member of the ClC family of chloride ion channels and antiporters, have been linked to Dent's disease, a renal disease associated with proteinuria. Several of the disease-causing mutations are premature stop mutations which lead to truncation of the C-terminus, pointing to the functional significance of this region. The C-terminus of ClC-5, like that of other eukaryotic ClC proteins, is cytoplasmic and contains a pair of CBS (cystathionine beta-synthase) domains connected by an intervening sequence. The presence of CBS domains implies a regulatory role for nucleotide interaction based on studies of other unrelated proteins bearing these domains [Ignoul and Eggermont (2005) Am. J. Physiol. Cell Physiol. 289, C1369-C1378; Scott, Hawley, Green, Anis, Stewart, Scullion, Norman and Hardie (2004) J. Clin. Invest. 113, 274-284]. However, to date, there has been no direct biochemical or biophysical evidence to support nucleotide interaction with ClC-5. In the present study, we have expressed and purified milligram quantities of the isolated C-terminus of ClC-5 (CIC-5 Ct). CD studies show that the protein is compact, with predominantly alpha-helical structure. We determined, using radiolabelled ATP, that this nucleotide binds the folded protein with low affinity, in the millimolar range, and that this interaction can be competed with 1 muM AMP. CD studies show that binding of these nucleotides causes no significant change in secondary structure, consistent with a model wherein these nucleotides bind to a preformed site. However, both nucleotides induce an increase in thermal stability of ClC-5 Ct, supporting the suggestion that both nucleotides interact with and modify the biophysical properties of this protein.