Many dramatic alterations in various cellular processes during the cell cycle are known to involve ion channels. In ascidian embryos and Caenorhabditis elegans oocytes, for example, the activity of inwardly rectifying Cl(-) channels is enhanced during the M phase of the cell cycle, but the mechanism underlying this change remains to be established. We show here that the volume-sensitive Cl(-) channel, ClC-2 is regulated by the M-phase-specific cyclin-dependent kinase, p34(cdc2)/cyclin B. ClC-2 channels were phosphorylated by p34(cdc2)/cyclin B in both in vitro and cell-free phosphorylation assays. ClC-2 phosphorylation was inhibited by olomoucine and abolished by a (632)Ser-to-Ala (S632A) mutation in the C-terminus, indicating that (632)Ser is a target of phosphorylation by p34(cdc2)/cyclin B. Injection of activated p34(cdc2)/cyclin B attenuated the ClC-2 currents but not the S632A mutant channel currents expressed in Xenopus oocytes. ClC-2 currents attenuated by p34(cdc2)/cyclin B were increased by application of the cyclin-dependent kinase inhibitor, olomoucine (100 microM), an effect that was inhibited by calyculin A (5 nM) but not by okadaic acid (5 nM). A yeast two-hybrid system revealed a direct interaction between the ClC-2 C-terminus and protein phosphatase 1. These data suggest that the ClC-2 channel is also counter-regulated by protein phosphatase 1. In addition, p34(cdc2)/cyclin B decreased the magnitude of ClC-2 channel activation caused by cell swelling. As the activities of both p34(cdc2)/cyclin B and protein phosphatase 1 vary during the cell cycle, as does cell volume, the ClC-2 channel could be regulated physiologically by these factors.

OBJECTIVE

The enteric nervous system integrates secretion and motility into homeostatic patterns of behavior susceptible to disorder. Progress in understanding mechanosensory detection in these processes, disordered enteric nervous system integration in diarrhea and constipation and pharmacotherapy is summarized.

RESULTS

Most neurons in the enteric nervous system discharge in response to distortion. Drugs acting directly to open chloride conductance channels in the mucosal epithelium are therapeutic options for constipation.

CONCLUSIONS

Mechanoreception is required for negative feedback control. At issue is identification of the neurons that fulfil the requirement for mechanoreception. Understanding secretomotor neurons is basic to understanding neurogenic secretory diarrhea and constipation and therapeutic strategies. A strategy for treatment of chronic constipation is development of agents that act directly to open Cl channels, which thereby increases the liquidity of the luminal contents. Lubiprostone, a recently Food and Drug Administration-approved drug, increases intraluminal liquidity by opening Cl channels. The future for the drug is clouded by controversy over whether its action is directly at one or the other of chloride channel type 2 (ClC-2) or cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels or both and whether action reflects involvement of G protein-coupled prostaglandin receptors expressed by mucosal epithelial cells.

In this Account, we describe the development of several diverse families of synthetic, membrane-active amphiphiles that form pores and facilitate transport within membrane bilayers. For the most part, the compounds are amphiphiles that insert into the bilayer and form pores either on their own or by self-assembly. The first family of synthetic ion channels prepared in our lab, the hydraphiles, used crown ethers as head groups and as a polar central element. In a range of biophysical studies, we showed that the hydraphiles formed unimolecular pores that spanned the bilayer. They mediated the transport of Na(+) and K(+) but were blocked by Ag(+). The hydraphiles are nonrectifying and disrupt ion homeostasis. As a result, these synthetic ion channels are toxic to various bacteria and yeast, a feature that has been used therapeutically in direct injection chemotherapy. We also developed a family of amphiphilic heptapeptide ion transporters that selected Cl(-) >10-fold over K(+) and showed voltage dependent gating. The formed pores were approximately dimeric, and variations in the N- and C-terminal anchor chains and the acids affected transport rates. Surprisingly, the longer N-terminal anchor chains led to less transport but greater Cl(-) selectivity. A proline residue, which is present in the ClC protein channel's conductance pore, proved to be critical for Cl(-) transport selectivity. Pyrogallol[4]arenes are macrocycles formed by acid-catalyzed condensation of four 1,2,3- trihydroxybenzenes with four aldehydes. The combination of 12 hydroxyl groups on one face of the macrocycle and four pendant alkyl chains conferred considerable amphiphilicity to these compounds. The pyrogallol[4]arenes inserted into bilayer membranes and conducted ions. Based on our experimental evidence, the ions passed through a self-assembled pore comprising four or five amphiphiles rather than passing through the central opening of a single macrocycle. Pyrogallol[4]arenes constructed with branched chains are also amphiphilic and active in membranes. The pyrogallol[4]arene with 3-pentyl sidechains formed a unique nanotube assembly and functioned as an ion channel in bilayer membranes. Finally, we showed that dianilides of either isophthalic or dipicolinic acids, compounds which have been extensively studied as anion binders, can self-assemble to form pores within bilayers. We called these dianilides tris-arenes and have shown that they readily bind to phosphate anions. These structures also mediated the transport of DNA plasmids through vital bilayer membranes in the bacterium Escherichia coli and in the yeast Saccharomyces cerevisiae . This transformation or transfection process occurred readily and without any apparent toxicity or mutagenicity.

The expression of several growth factors and K-ras gene mutation in bile were studied to better understand the pathogenesis and improve early diagnosis of bile duct cancers. Bile samples were collected from 12 cholangiocarcinomas (CLC), 10 ampullary cancers (APC), 3 gallbladder cancers (GBC), 7 pancreatic cancers (PNC), 9 biliary tract infection (BTI), 8 biliary stone disease (ST), and 5 normal controls (NC). The highest mean value of TGF-beta in bile was in patients with BTI; the mean levels of bFGF and PDGF were highest in CLC, and patients with APC and CLC had higher expression of HER2/Neu than other groups. In bile, a K-ras gene codon 12 mutation was found in 5 of 6 (83%) cases of CLC by the PCR-RFLP method. The results suggest overexpression of bFGF, PDGF, and HER2/Neu and the presence of K-ras mutation are important for carcinogenesis of bile duct cancers, and detection of the above abnormalities in bile is helpful for early diagnosis.

Recent evidence suggested that ClC-3 channel/antiporter is involved in regulation of nuclear factor (NF)-κB activation. However, the mechanism explaining how ClC-3 modulates NF-κB signaling is not well understood. We hypothesized that ClC-3-dependent alteration of intracellular chloride concentration ([Cl(-)](i)) underlies the effect of ClC-3 on NF-κB activity in endothelial cells. Here, we found that reduction of [Cl(-)](i) increased tumor necrosis factor-α (TNFα)-induced expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 and adhesion of monocytes to endothelial cells (P<0.05; n=6). In Cl(-) reduced solutions, TNFα-evoked IκB kinase complex β and inhibitors of κBα phosphorylation, inhibitors of κBα degradation, and NF-κB nuclear translocation were enhanced. In addition, TNFα and interleukin 1β could activate an outward rectifying Cl(-) current in human umbilical vein endothelial cells and mouse aortic endothelial cells. Knockdown or genetic deletion of ClC-3 inhibited or abolished this Cl(-) conductance. Moreover, Cl(-) channel blockers, ClC-3 knockdown or knockout remarkably reduced TNFα-induced intercellular adhesion molecule 1 and vascular cell adhesion molecule 1expression, monocytes to endothelial cell adhesion, and NF-κB activation (P<0.01; n=6). Furthermore, TNFα-induced vascular inflammation and neutrophil infiltration into the lung and liver were obviously attenuated in ClC-3 knockout mice (P<0.01; n=7). Our results demonstrated that decrease of [Cl(-)](i) induced by ClC-3-dependent Cl(-) efflux promotes NF-κB activation and thus potentiates TNFα-induced vascular inflammation, suggesting that inhibition of ClC-3-dependent Cl(-) current or modification of intracellular Cl(-) content may be a novel therapeutic approach for inflammatory diseases.

CLC-K chloride channels are expressed in the kidney, where they play a pivotal role in the mechanisms of urine concentration and Na(+) reabsorption. The identification of barttin as an essential beta-subunit of CLC-K channels allowed performance of a pharmacologic characterization of wild-type CLC-K1 expressed in Xenopus oocytes. To this end, a series of 2-(p-chlorophenoxy)propionic acid (CPP) derivatives were screened using the two-microelectrode voltage-clamp technique. Several chemical modifications regarding the phenoxy group of the side chain (elimination of the oxygen atom or of methylenic groups, substitutions of the chlorine atom) did not alter the drug blocking activity, with five different derivatives showing a similar potency. Among these, a derivative of CPP carrying a benzyl group on the chiral center in the place of the methyl group represented the minimal structure for blocking CLC-K1. It inhibited the channel from the extracellular side with an affinity in the 150 micro M range. The blocking potency of this compound is fourfold increased by lowering the extracellular chloride concentration, suggesting that the drug interacts with the channel pore. Concomitantly, the effect of some "classical" Cl(-) channel blockers (9-anthracenecarboxylic acid, 2-(phenylamino)benzoic acid, iminodibenzoic acid, niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt, and 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid disodium salt) was screened. 4,4'-Diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt was the only one capable of blocking CLC-K1 with a potency similar to the CPP derivative, although in an irreversible manner. The newly identified substances provide a useful tool to investigate the biophysical and physiologic role of these renal channels and a starting point for the development of therapeutic drugs with diuretic action.

We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLC-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages.

Huntington disease is a progressive and fatal genetic disorder with debilitating motor and cognitive defects. Chorea, rigidity, dystonia, and muscle weakness are characteristic motor defects of the disease that are commonly attributed to central neurodegeneration. However, no previous study has examined the membrane properties that control contraction in Huntington disease muscle. We show primary defects in ex vivo adult skeletal muscle from the R6/2 transgenic mouse model of Huntington disease. Action potentials in diseased fibers are more easily triggered and prolonged than in fibers from WT littermates. Furthermore, some action potentials in the diseased fibers self-trigger. These defects occur because of decreases in the resting chloride and potassium conductances. Consistent with this, the expression of the muscle chloride channel, ClC-1, in Huntington disease muscle was compromised by improper splicing and a corresponding reduction in total Clcn1 (gene for ClC-1) mRNA. Additionally, the total Kcnj2 (gene for the Kir2.1 potassium channel) mRNA was reduced in disease muscle. The resulting muscle hyperexcitability causes involuntary and prolonged contractions that may contribute to the chorea, rigidity, and dystonia that characterize Huntington disease.

The family of CLC proteins comprises both Cl(-) channels and Cl(-)/H(+) exchange transporters with varying degrees of voltage dependence. The human CLC-5 is an electrogenic voltage-dependent 2Cl(-)/1H(+) exchanger that gives rise to strongly outwardly rectifying currents when expressed. We conducted whole-cell recordings from HEK293 cells transiently transfected with either wild-type CLC-5 or a permeation-deficient mutant, E268A. With E268A CLC-5 we recorded transient voltage-dependent currents that represent the gating currents associated with CLC-5 activation and had kinetics that could be described by voltage-dependent forward and reverse transition rates. In extracellular solutions rich in Cl(-) or Br(-), CLC-5 exhibited a gating charge of 1.3, but this was reduced to 0.9 in solutions comprising the impermeant anions aspartate, methanesulfonate, sulfate, or HEPES. Extracellular ion depletion by local perfusion with isotonic mannitol failed to reduce the gating charge further. Lowering intracellular pH from 7.4 to 5.4 did not shift the voltage-dependence of the gating currents, but reducing and increasing intracellular Cl(-) shifted the charge-voltage relationship to more negative and positive potentials, respectively. Our data suggest that voltage sensing is an intrinsic property of the CLC-5 protein and that permeant anions, particularly Cl(-), modulate a voltage-dependent transition to an activated state from which Cl(-)/H(+) exchange can occur.

OBJECTIVE

The factors related to the learning curve for laparoscopic distal pancreatectomy have rarely been evaluated.

METHODS

A retrospective study of 32 patients who underwent a laparoscopic distal pancreatectomy performed at a high-volume center by a single pancreatic surgeon experienced with laparoscopic surgery was conducted. Pre-, intra- and postoperative data were collected. The primary endpoint was the length of the operation. The secondary endpoints were the conversion and reoperation rates, overall postoperative morbidity and mortality rates, the length of hospital stay and rate of unplanned splenectomy.

RESULTS

The length of the operation and the cumulative sum of the procedures presented a logarithmic correlation (P = 0.048). The learning curve appeared to have been completed after 17 procedures (P = 0.040). The multivariate analysis confirmed that the completion of the learning curve (CLC) reduced the length of the operation by 18 % (P = 0.009), but extended resection increased the length of the operation (P = 0.023). The conversion and reoperation rates, overall postoperative morbidity and mortality rates and length of the hospital stay were not related to the CLC. Unplanned splenectomy was more frequently performed during the first 17 procedures.

CONCLUSIONS

The length of the operation seems to be the main factor related to the CLC for laparoscopic distal pancreatectomy. The learning curve could be considered to be completed after about 17 procedures if performed by surgeons experienced with laparoscopic techniques at high-volume centers.

Rat cortical astrocytes regulate their cell volume in response to hypotonic challenge. This regulation is believed to depend largely on the release of chloride or organic osmolytes through anion channels. Using whole-cell recordings, we identified weakly outwardly rectifying chloride currents that could be activated in response to hypotonic challenge. These currents exhibited the following permeability sequence upon replacement of chloride in the bathing solution with various anions: I->> NO3->> Cl->> Gluc->> or = MeS->> Ise-. Interestingly, extracellular I-, albeit showing the greatest permeability, blocked the currents with an IC50 of approximately 50 mM. Currents were almost completely inhibited by 123 microM NPPB and partially inhibited by 200 microM niflumic acid or 200 microM DIDS. Additionally, the total number of Cl- ions effluxed through the hypotonically activated channels was markedly similar to the total solute efflux during volume regulation. We therefore propose the hypotonically activated chloride channel as a major contributor to volume regulation of astrocytes. To examine potential candidate chloride channel genes expressed by astrocytes, we employed RT-PCR to demonstrate the presence of transcripts for ClC-2, 3, 4, 5, and 7, as well as for VDAC and CFTR in cultured astrocytes. Moreover, we performed immunostaining with antibodies against each of these channels and showed the strongest expression of ClC-2 and ClC-3, strong expression of ClC-5 and VDAC, weak expression of ClC-7 and very weak expression of ClC-4 and CFTR. Intriguingly, although we found at least seven Cl- channel proteins from three different gene families in astrocytes, none appeared to be active in resting cells.

Proteins implicated as intracellular chloride channels include the intracellular ClC proteins, the bestrophins, the cystic fibrosis transmembrane conductance regulator, the CLICs, and the recently described Golgi pH regulator. This paper examines current hypotheses regarding roles of intracellular chloride channels and reviews the evidence supporting a role in intracellular chloride transport for each of these proteins.

We investigated the effect of truncations on the human muscle chloride channel CLC-1 and studied the functional complementation from partial proteins. Almost complete deletion of the cytoplasmic amino terminus did not affect currents, but truncating the intracellular COOH terminus after Leu720 abolished function. Currents were restored by coexpressing this membrane-embedded part with the lacking cytoplasmic fragment that contains domain D13, the second of the two conserved cystathionine beta-synthase (CBS) motifs present in all eukaryotic CLC proteins. However, if the cut was after Gln597 before the first CBS domain, no functional complementation was seen. Complementation was also obtained with channels "split" between transmembrane domains D7 and D8 or domains D8 and D9, but not when split between D10 and D11. Specificity of currents was tested by inserting point mutations in NH2-terminal (G188A and G230E) or COOH-terminal (K585E) fragments. In contrast to G188A and K585E, split channels did not tolerate the D136G mutation, suggesting that it may impede association from nonlinked fragments. Duplication, but not a lack of domain D8 was tolerated in "split" channels. Membrane domains D9-D12 can insert into the membrane without adding a preceding signal peptide to ensure the extracellular amino terminus of D9. Eventually, we succeeded in reconstituting CLC-1 channels from three separate polypeptides: the amino-terminal part up to D8, D9 through CBS1, and the remainder of the cytoplasmic carboxyl terminus. In summary, several regions of CLC channels behave autonomously regarding membrane insertion and folding and mediate protein-protein interactions strong enough to yield functional channels without a direct covalent link.

Cell shrinkage is an integral part of apoptosis. However, intimate mechanisms linking apoptotic events to the alterations in cell volume homeostasis remain poorly elucidated. We investigated how overexpression of Bcl-2 oncoprotein, a key antiapoptotic regulator, in lymph node carcinoma of the prostate (LNCaP) prostate cancer epithelial cells interferes with the volume-regulated anion channel (VRAC), a major determinant of regulatory volume decrease. Bcl-2 overexpression resulted in the doubling of VRAC-carried swelling-activated Cl(-) current (I(Cl,swell)) and weakened I(Cl,swell) inhibition by store-operated Ca(2+) channel (SOC)-transported Ca(2+). This also was accompanied by substantial up-regulation of ClC-3 protein, a putative molecular candidate for the role of VRAC. ClC-3-specific antibody suppressed I(Cl,swell) in the wild-type and Bcl-2-overexpressing LNCaP cells. Epidermal growth factor treatment of wild-type LNCaP cells, promoting their proliferation, resulted in the enhancement of endogenous Bcl-2 expression and associated increases in ClC-3 levels and I(Cl,swell) magnitude. We conclude that Bcl-2-induced up-regulation of I(Cl,swell), caused by enhanced expression of ClC-3 and weaker negative control from SOC-transported Ca(2+), would strengthen the ability of the cells to handle proliferative volume increases and thereby promote their survival and diminish their proapoptotic potential.

The chloride channel ClC-Kb is expressed in the basolateral cell membrane of the distal nephron and participates in renal NaCl reabsorption. Loss-of-function mutations of ClC-Kb lead to classic Bartter syndrome, a rare salt-wasting disorder. Recently, we identified the ClC-Kb(T481S) polymorphism, which confers a strong gain-of-function effect on the ClC-Kb chloride channel. The present study has been performed to explore the prevalence of the mutation and its functional significance in renal salt handling and blood pressure regulation. As evident from electrophysiological analysis with the 2-electrode voltage-clamp technique, heterologous expression of ClC-Kb(T481S) in Xenopus oocytes gave rise to a current that was 7-fold larger than the current produced by wild-type ClC-Kb. The prevalence of the mutant allele was significantly higher in an African population from Ghana (22%) than in whites (12%). As tested in 1 white population, carriers of ClC-Kb(T481S) were associated with significantly higher systolic (by approximately 6.0 mm Hg) and diastolic (by approximately 4.2 mm Hg) blood pressures and significantly higher prevalence (45% versus 25%) of hypertensive >> or =140/90 mm Hg) blood pressure levels. Individuals carrying ClC-Kb(T481S) had significantly higher plasma Na+ concentrations and significantly decreased glomerular filtration rate. In conclusion, the mutation ClC-Kb(T481S) of the renal epithelial Cl- channel ClC-Kb strongly activates ClC-Kb chloride channel function in vitro and may predispose to the development of essential hypertension in vivo.

It has been previously determined that ClC-2, a member of the ClC chloride channel superfamily, is expressed in certain epithelial tissues. These findings fueled speculation that ClC-2 can compensate for impaired chloride transport in epithelial tissues affected by cystic fibrosis and lacking the cystic fibrosis transmembrane conductance regulator. However, direct evidence linking ClC-2 channel expression to epithelial chloride secretion was lacking. In the present studies, we show that ClC-2 transcripts and protein are present endogenously in the Caco-2 cell line, a cell line that models the human small intestine. Using an antisense strategy we show that ClC-2 contributes to native chloride currents in Caco-2 cells measured by patch clamp electrophysiology. Antisense ClC-2-transfected monolayers of Caco-2 cells exhibited less chloride secretion (monitored as iodide efflux) than did mock transfected monolayers, providing the first direct molecular evidence that ClC-2 can contribute to chloride secretion by the human intestinal epithelium. Further, examination of ClC-2 localization by confocal microscopy revealed that ClC-2 contributes to secretion from a unique location in this epithelium, from the apical aspect of the tight junction complex. Hence, these studies provide the necessary rationale for considering ClC-2 as a possible therapeutic target for diseases affecting intestinal chloride secretion such as cystic fibrosis.

The genetic structure of two Ralstonia spp., strain JS705 and strain JS745, isolated from the same groundwater aquifer, was characterized with respect to the degradation capacities for toluene and chlorobenzene degradation. Cosmid library construction, cloning, DNA sequencing and mating experiments indicated that the genes for chlorobenzene degradation in strain JS705 were a mosaic of the clc genes, previously described for Pseudomonas sp. strain B13, and a 5 kb fragment identical to strain JS745. The 5 kb fragment identical to both JS705 and JS745 was flanked in JS705 by one complete and one incomplete insertion (IS) element. This suggested involvement of the IS element in mobilizing the genes from JS745 to JS705, although insertional activity of the IS element in its present configuration could not be demonstrated. The complete genetic structure for chlorobenzene degradation in strain JS705 resided on a genomic island very similar to the clc element (Ravatn, R., Studer, S., Springael, D., Zehnder, A.J., van der Meer, J.R. 1998. Chromosomal integration, tandem amplification, and deamplification in Pseudomonas putida F1 of a 105-kilobase genetic element containing the chlorocatechol degradative genes from Pseudomonas sp. strain B13. J Bacteriol 180: 4360-4369). The unique reconstruction of formation of a metabolic pathway through the activity of IS elements and a genomic island in the chlorobenzene-degrading strain JS705 demonstrated how pathway evolution can occur under natural conditions in a few 'steps'.

Channels and transporters of the ClC family serve a variety of physiological functions. Understanding of their gating and transport mechanisms remains incomplete, with disagreement over the extent of protein conformational change involved. Using site-directed fluorescence labeling, we probe ClC-ec1, a prokaryotic ClC, for transport-related structural rearrangements. We specifically label cysteines introduced at several positions in the R helix of ClC-ec1 with AlexaFluor 488, an environment-sensitive fluorophore, and demonstrate that the labeled mutants show H+/Cl- transport activity indistinguishable from that of the wild-type protein. At each position that we examined we observe fluorescence changes upon acidification over the same pH range that is known to activate transport. The fluorescence change is also sensitive to Cl- concentration; furthermore, the Cl- and H+ dependencies are coupled as would be expected if the fluorescence change reflected a conformational change required for transport. Together, the results suggest that the changes in fluorescence report protein conformational changes underlying the transport process. Labeled transporters mutated to remove a glutamate critical to proton-coupled chloride transport retain pH-dependent fluorescence changes, suggesting that multiple residues confer pH dependence on the transport mechanism. These results have implications for models of transport and gating in ClC channels and transporters.

The skeletal muscle-specific ClC-1 is a voltage-gated chloride channel protein. Specific antibodies against ClC-1 revealed in muscle sections a sarcolemmal staining that was absent in the myotonic arrested development of righting response (ADR) mouse muscle. The intensity of the sarcolemmal staining varied from one type of muscle to another and in lateral sections showed a typical mosaic pattern that colocalized with beta-dystroglycan and left the transverse tubule openings clear. Surprisingly, in isolated myofibers, the ClC-1 protein was absent from the sarcolemma. Instead, it localized to intracellular I band areas as soon as the myofibers were isolated. When the isolated myofibers were incubated with the kinase inhibitor staurosporine, the ClC-1 protein shifted back to the sarcolemma. Electric stimulation of the cultivated fibers had a similar effect. Also, myofibers infected with a recombinant Semliki Forest virus (SFV) expressing myc-tagged ClC-1 showed intracellular localization of the protein. The virally expressed mycClC-1 reached the Golgi apparatus but sarcolemmal staining remained nondetectable, and addition of staurosporine into the growth medium recruited the mycClC-1 to the sarcolemma. These data indicate that sarcolemmal targeting of the ClC-1 requires specific signals that are provided by the physiological environment.

Characterization of the human blood plasma proteome is critical to the discovery of routinely useful clinical biomarkers. We used an accurate mass and time (AMT) tag strategy with high-resolution mass accuracy cLC-FT-ICR MS to perform a global proteomic analysis of pilot study samples as part of the HUPO Plasma Proteome Project. HUPO reference serum and citrated plasma samples from African Americans, Asian Americans, and Caucasian Americans were analyzed, in addition to a Pacific Northwest National Laboratory reference serum and plasma. The AMT tag strategy allowed us to leverage two previously published "shotgun" proteomics experiments to perform global analyses on these samples in triplicate in less than 4 days total analysis time. A total of 722 (22% with multiple peptide identifications) International Protein Index redundant proteins, or 377 protein families by ProteinProphet, were identified over the six individual HUPO serum and plasma samples. The samples yielded a similar number of identified redundant proteins in the plasma samples (average 446 +/- 23) as found in the serum samples (average 440 +/- 20). These proteins were identified by an average of 956 +/- 35 unique peptides in plasma and 930 +/- 11 unique peptides in serum. In addition to this high-throughput analysis, the AMT tag approach was used with a Z-score normalization to compare relative protein abundances. This analysis highlighted both known differences in serum and citrated plasma such as fibrinogens, and reproducible differences in peptide abundances from proteins such as soluble activin receptor-like kinase 7b and glycoprotein m6b. The AMT tag strategy not only improved our sample throughput but also provided a basis for estimated quantitation.

Regiospecific cyclizations of the nascent poly-beta-ketone backbones dictate the structures of polyketide natural products. The fungal iterative megasynthases use terminal thioesterase/claisen cyclase (TE/CLC) domains to direct the fate of the polyketide chains. In this work, we present two strategies toward redirecting the cyclization steps of fungal PKSs using the Gibberella fujikuroi PKS4. First, inactivation or removal of the TE/CLC domain resulted in the synthesis of the new polyketide SMA93 2. Complementation of the mutant PKS4 with a standalone TE/CLC domain restored the regioselective cyclization steps of PKS4 and led to the synthesis of SMA76 1, demonstrating that cyclization enzymes can interact with the megasynthase in trans. This led to the second approach in which various dissociated, bacterial tailoring enzymes were added to the megasynthase in trans. Addition of the act KR led to the synthesis of mutactin 3, while the addition of first ring and second ring cyclases yielded anthraquinone compounds DMAC 5 and SEK26 6. The cooperative activities of fungal and bacterial PKS components are especially important and enable synthesis of polyketides utilizing enzymes from two distinct families of PKSs.

1. Gating of the skeletal muscle chloride channel (ClC-1) is sensitive to extracellular pH. In this study, whole-cell recording of currents from wild-type (WT) ClC-1 and a mutant, R304E, expressed in the Sf-9 insect cell line was used to investigate further the nature of the pH-sensitive residues. 2. Extracellular Cd2+ produced a concentration-dependent block of WT ClC-1 with an IC50 of 1.0 +/- 0.1 mM and a Hill coefficient of 2.0 +/- 0.3. This block was sensitive to external pH, reducing at low pH, with an apparent pKa of 6.8 +/- 0.1 and a Hill coefficient for proton binding of 3.0 +/- 0.3. Anthracene-9-carboxylate (A-9-C) block of WT ClC-1 was also pH sensitive, increasing at low pH, with an apparent pKa of 6.4 +/- 0.1 and a Hill coefficient for proton binding of 1.0 +/- 0.2. 3. Compared with WT ClC-1, R304E had a lower affinity for Cd2+ (IC50, 3.0 +/- 0.3 mM) but it had a similar Hill coefficient for transition metal ion binding. The Hill coefficient for proton binding to the Cd2+ binding site was reduced to 1.4 +/- 0.3. In contrast, the A-9-C binding site in R304E showed the same pH sensitivity and affinity for the blocker as that seen in WT ClC-1. 4. ClC-1 has at least two binding sites for Cd2+, each of which has at least three residues which can be protonated. Binding of A-9-C is influenced by protonation of a single residue. Arg 304 is not sufficiently close to the A-9-C binding site to affect its characteristics, but it does. alter Cd2+ binding, indicating that transition metal ions and aromatic carboxylates interact with distinct sites. 5. The block of ClC-1 by transition metal ions and the apparent pKa of this block, together with the apparent pKa for A-9-C block and gating are all compatible with the involvement of His residues in the pore and gate of ClC-1.

Activation of the CFTR Cl(-) channel inhibits epithelial Na(+) absorption, according to studies on native epithelia derived from airways, colon and kidney, and can also be demonstrated in overexpressing cells. However, Na(+) absorption is not inhibited by CFTR in the native sweat duct epithelium. The mechanism for the inhibition of epithelial sodium channels (ENaC) has been examined in most detail in Xenopus oocytes coexpressing CFTR and ENaC. It was shown that ENaC is inhibited during stimulation of CFTR in Xenopus oocytes, independent of the experimental setup and the magnitude of the whole-cell current. However, a minimal Cl(-) conductance is required for inhibition of ENaC, and inhibition is augmented at higher CFTR-to-ENaC currents ratios. Low-CFTR-to-ENaC conductance ratios may be the reason for the absence of ENaC inhibition, as described recently. Similar to CFTR, ClC-0 Cl(-) currents also inhibit ENaC, as well as high extracellular Na(+) and Cl(-) in partially permeabilized oocytes. Thus, inhibition of ENaC is not specific to CFTR and could be mediated by Cl(-) flow and/or changes in the intracellular Cl(-) concentration. These results are reminiscent of the Cl(-) feedback regulation observed in mouse mandibular duct cells. Current results obtained with ENaC mutants examined in Xenopus oocytes suggest a charge interaction of Cl(-) ions with the epithelial sodium channel.

Changes in phosphorylation regulate the activity of various ClC anion transport proteins. However, the physiological context under which such regulation occurs and the signaling cascades that mediate phosphorylation are poorly understood. We have exploited the genetic model organism Caenorhabditis elegans to characterize ClC regulatory mechanisms and signaling networks. CLH-3b is a ClC anion channel that is expressed in the worm oocyte and excretory cell. Channel activation occurs in response to oocyte meiotic maturation and swelling via serine/threonine dephosphorylation mediated by the type I phosphatases GLC-7alpha and GLC-7beta. A Ste20 kinase, germinal center kinase (GCK)-3, binds to the cytoplasmic C terminus of CLH-3b and inhibits channel activity in a phosphorylation-dependent manner. Analysis of hyperpolarization-induced activation kinetics suggests that phosphorylation may inhibit the ClC fast gating mechanism. GCK-3 is an ortholog of mammalian SPAK and OSR1, kinases that bind to, phosphorylate, and regulate the cell volume-dependent activity of mammalian cation-Cl(-) cotransporters. Using mass spectrometry and patch clamp electrophysiology, we demonstrate here that CLH-3b is a target of regulatory phosphorylation. Concomitant phosphorylation of S742 and S747, which are located 70 and 75 amino acids downstream from the GCK-3 binding site, are required for kinase-mediated channel inhibition. In contrast, swelling-induced channel activation occurs with dephosphorylation of S747 alone. Replacement of both S742 and S747 with glutamate gives rise to kinase- and swelling-insensitive channels that exhibit activity and biophysical properties similar to those of wild-type CLH-3b inhibited by GCK-3. Our studies provide novel insights into ClC regulation and mechanisms of cell volume signaling, and provide the foundation for studies aimed at defining how conformational changes in the cytoplasmic C terminus alter ClC gating and function in response to intracellular signaling events.