Channels and transporters of the ClC family cause the transmembrane movement of inorganic anions in service of a variety of biological tasks, from the unusual-the generation of the kilowatt pulses with which electric fish stun their prey-to the quotidian-the acidification of endosomes, vacuoles and lysosomes. The homodimeric architecture of ClC proteins, initially inferred from single-molecule studies of an elasmobranch Cl(-) channel and later confirmed by crystal structures of bacterial Cl(-)/H(+) antiporters, is apparently universal. Moreover, the basic machinery that enables ion movement through these proteins-the aqueous pores for anion diffusion in the channels and the ion-coupling chambers that coordinate Cl(-) and H(+) antiport in the transporters-are contained wholly within each subunit of the homodimer. The near-normal function of a bacterial ClC transporter straitjacketed by covalent crosslinks across the dimer interface and the behaviour of a concatemeric human homologue argue that the transport cycle resides within each subunit and does not require rigid-body rearrangements between subunits. However, this evidence is only inferential, and because examples are known in which quaternary rearrangements of extramembrane ClC domains that contribute to dimerization modulate transport activity, we cannot declare as definitive a 'parallel-pathways' picture in which the homodimer consists of two single-subunit transporters operating independently. A strong prediction of such a view is that it should in principle be possible to obtain a monomeric ClC. Here we exploit the known structure of a ClC Cl(-)/H(+) exchanger, ClC-ec1 from Escherichia coli, to design mutants that destabilize the dimer interface while preserving both the structure and the transport function of individual subunits. The results demonstrate that the ClC subunit alone is the basic functional unit for transport and that cross-subunit interaction is not required for Cl(-)/H(+) exchange in ClC transporters.

Bartter's and Gitelman's syndromes are characterized by hypokalemia, normal to low blood pressure and hypochloremic metabolic alkalosis. Recently, investigators have been able to demonstrate mutations of six genes encoding several renal tubular transporters and ion channels that can be held responsible for Bartter's and Gitelman's syndromes. Neonatal Bartter's syndrome is caused by mutations of NKCC2 or ROMK, classic Bartter's syndrome by mutations of ClC-Kb, Bartter's syndrome associated with sensorineural deafness is due to mutations of BSND, Gitelman's syndrome to mutations of NCCT and Bartter's syndrome associated with autosomal dominant hypocalcemia is linked to mutations of CASR. We review the pathophysiology of these syndromes in relation to their clinical presentation.

ClC-3 is a voltage-gated Cl- channel that is highly conserved and widely expressed, although its function, localization, and properties remain a matter of considerable debate. In this study, we have shown that heterologous expression of ClC-3 in either Chinese hamster ovary (CHO-K1) or human hepatoma (Huh-7) cells results in the formation of large, acidic vesicular structures within cells. Vesicle formation is prevented by bafilomycin, an inhibitor of the vacuolar ATPase, and is not induced by an E224A mutant of ClC-3 with altered channel activity. This demonstrates that vesicle formation requires both proton pumping and Cl- channel activity. Manipulation of the intracellular Cl- concentration demonstrated that the ClC-3-associated vesicles shrink and swell consistent with a highly Cl--permeable membrane. The ClC-3 vesicles were identified as lysosomes based on their colocalization with the lysosome-associated proteins lamp-1, lamp-2, and cathepsin D and on their failure to colocalize with fluorescently labeled endosomes. We conclude that ClC-3 is an intracellular channel that conducts Cl- when it is present in intracellular vesicles. Its overexpression results in its appearance in enlarged lysosome-like structures where it contributes to acidification by charge neutralization.

Gitelman syndrome is an inherited renal disorder characterized by impaired NaCl reabsorption in the distal convoluted tubule and secondary hypokalemic alkalosis. In clinical practice, it is distinguished from other hypokalemic tubulopathies by the presence of both hypomagnesemia and normocalcemic hypocalciuria. To date, only mutations in a single gene encoding the thiazide-sensitive NaCl cotransporter have been found as the molecular basis of GS. We describe three unrelated patients presenting with the typical laboratory findings of GS. Mutational analysis in these patients revealed no abnormality in the SLC12A3 gene. Instead, all patients were found to carry previously described mutations in the CLCNKB gene, which encodes the kidney-specific chloride channel ClC-Kb, raising the possibility of genetic heterogeneity. Review of the medical histories revealed manifestation of the disease within the first year of life in all cases. Clinical presentation included episodes of dehydration, weakness, and failure to thrive, much more suggestive of classic Bartter syndrome than of GS. The coexistence of hypomagnesemia and hypocalciuria was not present from the beginning. In the follow-up, however, a drop of both parameters below normal range was a consistent finding reflecting a transition from cBS to GS phenotype. The phenotypic overlap may indicate a physiologic cooperation of the apical thiazide-sensitive NaCl cotransporter and the basolateral chloride channel for salt reabsorption in the distal convoluted tubule.

The Cl- channel ClC-2 is expressed in transporting epithelia and has been proposed as an alternative route for Cl- efflux that might compensate for the malfunction of CFTR in cystic fibrosis. There is controversy concerning the cellular and membrane location of ClC-2, particularly in intestinal tissue. The aim of this paper is to resolve this controversy by immunolocalization studies using tissues from ClC-2 knockout animals as control, ascertaining the sorting of ClC-2 in model epithelial cells and exploring the possible molecular signals involved in ClC-2 targeting. ClC-2 was exclusively localized at the basolateral membranes of surface colonic cells or villus duodenal enterocytes. ClC-2 was sorted to the basolateral membranes in MDCK, Caco-2 and LLC-PK1-mu1B, but not in LLC-PK1-mu1A cells. Mutating a di-leucine motif (L812L813) to a di-alanine changed the basolateral targeting of ClC-2 to an apical location. The basolateral membrane localization of ClC-2 in absorptive cells of the duodenum and the colon is compatible with an absorptive function for this Cl- channel. Basolateral targeting information is contained in a di-leucine motif (L812L813) within CBS-2 domain at the C-terminus of ClC-2. It is speculated that ClC-2 also contains an apical sorting signal masked by L812L813. The proposal that CBS domains in ClC channels might behave as regulatory sites sensing intracellular signals opens an opportunity for pharmacological modulation of ClC-2 targeting.

OBJECTIVE

Lowe syndrome is defined by congenital cataracts, mental retardation, and proximal tubulopathy and is due to mutations in OCRL. Recently, mutations in OCRL were found to underlie some patients with Dent disease, characterized by low molecular weight proteinuria, hypercalciuria, and nephrocalcinosis. This phenotypic heterogeneity is poorly understood.

METHODS

The renal phenotype of 16 patients with Lowe syndrome (10.9 +/- 7.0 yr) under care of the authors was characterized to define overlap of symptoms with Dent disease and infer clues about OCRL function. Medical charts of patients were reviewed for data regarding glomerular filtration rate and markers of proximal tubular function.

RESULTS

All patients had low molecular weight proteinuria and albuminuria. Lysosomal enzymuria was elevated in all 11 patients assessed. Fifteen patients had hypercalciuria, and 14 aminoaciduria. Seven patients required bicarbonate and three required phosphate replacement; all others maintained normal serum values without supplementation. None of the patients had detectable glycosuria, and none had clinically overt rickets. GFR was mildly to moderately impaired and highly variable, with a trend of deterioration with age.

CONCLUSIONS

Patients with Lowe syndrome do not have renal Fanconi syndrome but a selective proximal tubulopathy, variable in extent and dominated by low molecular weight proteinuria and hypercalciuria, the classical features of Dent disease. These findings suggest that OCRL and ClC-5, the chloride channel mutated in Dent disease, are involved in similar reabsorption pathways in the proximal tubule.

BACKGROUND

One of the most common symptoms among patients with inflammatory bowel disease (IBD) is diarrhea, which is thought to be contributed by changes in electrolyte transport associated with intestinal inflammation. This study was designed to test the hypothesis that intestinal Na(+)-related transporters/channels and their regulatory proteins may be downregulated as a potential contributor to IBD-associated diarrhea.

METHODS

SDS-PAGE and Western blotting and/or confocal immunomicroscopy were used to examine the expression of Na(+)/H(+)-exchangers 1-3 (NHE1-3), epithelial Na(+) channel (ENaC), Na(+)/K(+)-ATPase, the intracellular Cl(-) channel 5 (ClC-5), and NHE3 regulatory factors (NHERF1,2) in ileal and colonic pinch biopsies from IBD patients and noninflammatory controls, as well as from colonic mucosa of dextran sodium sulfate (DSS)- and TNBS-induced acute murine IBD models.

RESULTS

NHE1,3 (but not NHE2), beta-ENaC, Na(+)/K(+)-ATPase-alpha, ClC-5, and NHERF1 were all downregulated in sigmoid mucosal biopsies from most cases of active UC and/or CD compared to controls. NHE3 was also decreased in ileal mucosal biopsies of active CD, as well as in approximately 50% of sigmoid biopsies from inactive UC or CD. Importantly, similar downregulation of NHE1,3, beta-ENaC, and NHERF1,2 was also observed in the mouse colon (but not ileum) of DSS- and TNBS-induced colitis.

CONCLUSIONS

IBD-associated diarrhea may be due to a coordinated downregulation of multiple Na(+) transporter and related regulatory proteins, including NHE1,3, Na(+)/K(+)-ATPase, and ENaC, as well as NHERF1,2, and ClC-5, all of which are involved directly or indirectly in intestinal Na(+) absorption.

We have cloned four novel members of the CLC family of chloride channels from Arabidopsis thaliana. The four plant genes are homologous to a recently isolated chloride channel gene from tobacco (CLC-Nt1; Lurin, C., Geelen, D., Barbier-Brygoo, H., Guern, J., and Maurel, C. (1996) Plant Cell 8, 701-711) and are about 30% identical in sequence to the most closely related CLC-6 and CLC-7 putative chloride channels from mammalia. AtCLC transcripts are broadly expressed in the plant. Similarly, antibodies against the AtCLC-d protein detected the protein in all tissues, but predominantly in the silique. AtCLC-a and AtCLC-b are highly homologous to each other ( approximately 87% identity), while being approximately 50% identical to either AtCLC-c or AtCLC-d. None of the four cDNAs elicited chloride currents when expressed in Xenopus oocytes, either singly or in combination. Among these genes, only AtCLC-d could functionally substitute for the single yeast CLC protein, restoring iron-limited growth of a strain disrupted for this gene. Introduction of disease causing mutations, identified in human CLC genes, abolished this capacity. Consistent with a similar function of both proteins, the green fluorescent protein-tagged AtCLC-d protein showed the identical localization pattern as the yeast ScCLC protein. This suggests that in Arabidopsis AtCLC-d functions as an intracellular chloride channel.

Regulated secretion in exocrine and neuroendocrine cells occurs through exocytosis of secretory granules and the subsequent release of stored small molecules and proteins. The introduction of biophysical techniques with high temporal and spatial resolution, and the identification of Ca(2+)-dependent and -independent "docking" and "fusion" proteins, has greatly enhanced our understanding of exocytosis. The cloning of families of ion channel proteins, including intracellular ion channels, has also revived interest in the role of secretory granule ion channels in exocytotic secretion. Thus secretory granules of pancreatic acinar cell express a ClC-2 Cl(-) channel, a HCO-permeable member of the CLCA Ca(2+)-dependent anion channel family, and a KCNQ1 K(+) channel. Evidence suggests that these channels may facilitate the release of digestive enzymes and/or prevent exocytosed granules from collapsing during "kiss and run" recycling. In pancreatic beta-cells, a granular ClC-3 Cl(-) channel provides a shunt pathway for a vacuolar-type H(+)-ATPase. Acidification "primes" the granules for Ca(2+)-dependent exocytosis and release of insulin. In summary, secretory granules are equipped with specific sets of ion channels, which modulate regulated exocytosis and the release of macromolecules. These channels could represent excellent targets for therapeutic interventions to control exocytotic secretion in relevant diseases, such as pancreatitis, cystic fibrosis, or diabetes mellitus.

Voltage-gated ClC chloride channels play important roles in cell volume regulation, control of muscle excitability, and probably transepithelial transport. ClC channels can be functionally expressed without other subunits, but it is unknown whether they function as monomers. We now exploit the properties of human mutations in the muscle chloride channel, ClC-1, to explore its multimeric structure. This is based on analysis of the dominant negative effects of ClC-1 mutations causing myotonia congenita (MC, Thomsen's disease), including a newly identified mutation (P480L) in Thomsen's own family. In a co-expression assay, Thomsen's mutation dramatically inhibits normal ClC-1 function. A mutation found in Canadian MC families (G230E) has a less pronounced dominant negative effect, which can be explained by functional WT/G230E heterooligomeric channels with altered kinetics and selectivity. Analysis of both mutants shows independently that ClC-1 functions as a homooligomer with most likely four subunits.

OBJECTIVE

Lubiprostone (Amitiza), a possible ClC-2 channel opener derived from prostaglandin E(1) and indicated for the treatment of constipation, increases chloride ion transport and fluid secretion into the intestinal lumen. As lubiprostone may also directly modulate gastrointestinal motility, we investigated its actions and the possible involvement of prostaglandin EP receptor activation on rat and human isolated gastrointestinal preparations.

METHODS

Rat and human isolated preparations were mounted in tissue baths for isometric recording. The effects of lubiprostone on muscle tension and on electrically stimulated, neuronal contractions were investigated in the absence and presence of EP receptor antagonists.

RESULTS

In rat and human stomach longitudinal muscle, lubiprostone induced a contraction (pEC(50) of 7.0+/-0.0, n=4 and 6.4+/-0.2, n=3, respectively), which was inhibited by pretreatment with the EP(1) receptor antagonist, EP(1)A 300 nM (pEC(50) reduced to 6.2+/-0.2, n=6), but not by the EP(3) or EP(4) receptor antagonists (L-798106 and GW627368X, respectively, 1 microM, P>0.05). Lubiprostone also reduced electrically stimulated, neuronal contractions in rat and human colon circular muscle preparations (pIC(50) of 8.9+/-0.4, n=7 and 8.7+/-0.9, n=6, respectively), an effect mediated pre-junctionally. This effect was reduced by the EP(4) receptor antagonist (pIC(50) of 6.7+/-1.1, n=7 and 7.7+/-0.4, n=6, respectively) but not by EP(1) or EP(3) receptor antagonists.

CONCLUSIONS

In rats and humans, lubiprostone contracts stomach longitudinal muscle and inhibits neuronally mediated contractions of colon circular muscle. Experiments are now needed to determine if this additional activity of lubiprostone contributes to its clinical efficacy and/or side-effect profile.

The Torpedo Cl- channel, CLC-0, is inhibited by clofibric acid derivatives from the intracellular side. We used the slow gate-deficient mutant CLC-0C212S to investigate the mechanism of block by the clofibric acid-derivative p-chlorophenoxy-acetic acid (CPA). CPA blocks open channels with low affinity (KDO= 45 mM at 0 mV) and shows fast dissociation (koff = 490 s-1 at -140 mV). In contrast, the blocker binds to closed channels with higher affinity and with much slower kinetics. This state-dependent block coupled with the voltage dependence of the gating transitions results in a highly voltage-dependent inhibition of macroscopic currents (KD approximately 1 mM at -140 mV; KD approximately 65 mM at 60 mV). The large difference in CPA affinity of the open and closed state suggests that channel opening involves more than just a local conformational rearrangement. On the other hand, in a recent work (Dutzler, R., E.B. Campbell, and R. MacKinnon. 2003. Science. 300:108-112) it was proposed that the conformational change underlying channel opening is limited to a movement of a single side chain. A prediction of this latter model is that mutations that influence CPA binding to the channel should affect the affinities for an open and closed channel in a similar manner since the general structure of the pore remains largely unchanged. To test this hypothesis we introduced point mutations in four residues (S123, T471, Y512, and K519) that lie close to the intracellular pore mouth or to the putative selectivity filter. Mutation T471S alters CPA binding exclusively to closed channels. Pronounced effects on the open channel block are observed in three other mutants, S123T, Y512A, and K519Q. Together, these results collectively suggest that the structure of the CPA binding site is different in the open and closed state. Finally, replacement of Tyr 512, a residue directly coordinating the central Cl- ion in the crystal structure, with Phe or Ala has very little effect on single channel conductance and selectivity. These observations suggest that channel opening in CLC-0 consists in more than a movement of a side chain and that other parts of the channel and of the selectivity filter are probably involved.

Guinea pig eosinophil granules contain a protein, the major basic protein (MBP), which accounts for more than half of the total granule protein, has a high content of arginine, and displays a remarkable tendency to form disulfide-linked aggregates. In this study we have purified a similar protein from human eosinophil granules and have compared the human MBP to the protein comprising the Charcot-Leyden crystal (CLC). Eosinophils from patients with various diseases were purified and disrupted, and the granule fraction was obtained. Examination of the granule fraction by transmission electron microscopy showed numerous typical eosinophil granules. Analyses of granule lysates by gel filtration and by polyacrylamide gel electrophoresis revealed the presence of peroxidase and MBP with properties similar to that previously found in guinea pig eosinophil granules. The human MBP had a molecular weight of 9,200, contained less than 1% carbohydrate, was rich in arginine, and readily formed disulfide-bonded aggregates. CLC were prepared from eosinophil-rich cell suspensions by homogenization in hypotonic saline. The supernates following centrifugation of cell debris spontaneously formed CLC. Analysis of CLC revealed the presence of a protein with a molecular weight of 13,000 containing 1.2% carbohydrate. The protein displayed a remarkable tendency to aggregate even in the presence of 0.2 M acetic acid. Human MBP and CLC protein differed in their molecular weights, carbohydrate compositions, and amino acid analyses. Mixtures of the MBP and the CLC protein yielded two bands in polyacrylamide gel electrophoresis. Neither eosinophil protein increased vascular permeability in the guinea pig skin or contracted the guinea pig ileum. The results indicate that the human MBP and the CLC are distinct substances with properties such that one cannot be derived from the other.

Recruitment of acid hydrolases to lysosomes generally occurs by intracellular sorting based on recognition of a common mannose 6-phosphate signal in the transGolgi network and selective transport to late endosomes/lysosomes. Here we provide evidence for an alternative, efficient secretion-recapture pathway mediated by megalin and exemplified by cathepsin B in kidney proximal convoluted tubules (PCT). We found that in mouse kidneys with defective megalin expression [megalin knockout (KO)] or apical PCT trafficking (ClC-5 KO), the (pro)cathepsin B mRNA level was essentially preserved, but the protein content was greatly decreased and the enzyme was excreted in the urine as mannose 6-phosphate-devoid species. In polarized PCT-derived cells, purified cathepsin B was avidly and selectively taken up at the apical membrane, and uptake was abolished by the megalin competitor, receptor-associated protein. Direct interaction of cathepsin B with megalin was demonstrated by surface plasmon resonance. Procathepsin B was detected in normal mouse serum. Purified cathepsin B injected into mice was efficiently taken up by kidneys (approximately 10% of injection) and targeted to lysosomes where it remained active, as shown by autoradiography and subcellular fractionation. A single cathepsin B injection into cathepsin B KO mice could reconstitute full lysosomal enzyme activity in the kidneys. These findings demonstrate a pathway whereby circulating lysosomal enzymes are continuously filtered in glomeruli, reabsorbed by megalin-mediated endocytosis, and transferred into lysosomes to exert their function, providing a major source of enzymes to PCT. These results also extend the significance of megalin in PCT and have several physiopathological and clinical implications.

Next-generation sequencing enables the study of species without a sequenced genome at the 'omics' level. Custom transcriptome databases are generated and global expression profiles can be compared. However, the assembly of transcriptome sequence reads into contigs remains a daunting task. In this study, five different assembly programs, both traditional overlap-based, 'read-centric' assemblers and de Bruijn graph data structure-based assemblers, were compared. To this end, artificial read libraries with and without simulated sequencing errors were constructed from Arabidopsis thaliana, based on quantitative profiles of mature leaf tissue. The open source TGICL pipeline and the commercial CLC bio genomics workbench produced the best assemblies in terms of contig length, hybrid assemblies, redundancy reduction, and error tolerance. The mature leaf transcriptomes of the C(3) species Cleome spinosa and the C(4) species Cleome gynandra were assembled and analysed. The pathways and cellular processes tagged in the transcriptome assemblies reflect processes of a mature leaf. The databases are useful for extracting transcripts related to C(4) processes as full-length or nearly full-length sequences.

BACKGROUND

Molecular breeding of pepper (Capsicum spp.) can be accelerated by developing DNA markers associated with transcriptomes in breeding germplasm. Before the advent of next generation sequencing (NGS) technologies, the majority of sequencing data were generated by the Sanger sequencing method. By leveraging Sanger EST data, we have generated a wealth of genetic information for pepper including thousands of SNPs and Single Position Polymorphic (SPP) markers. To complement and enhance these resources, we applied NGS to three pepper genotypes: Maor, Early Jalapeño and Criollo de Morelos-334 (CM334) to identify SNPs and SSRs in the assembly of these three genotypes.

RESULTS

Two pepper transcriptome assemblies were developed with different purposes. The first reference sequence, assembled by CAP3 software, comprises 31,196 contigs from >125,000 Sanger-EST sequences that were mainly derived from a Korean F1-hybrid line, Bukang. Overlapping probes were designed for 30,815 unigenes to construct a pepper Affymetrix GeneChip® microarray for whole genome analyses. In addition, custom Python scripts were used to identify 4,236 SNPs in contigs of the assembly. A total of 2,489 simple sequence repeats (SSRs) were identified from the assembly, and primers were designed for the SSRs. Annotation of contigs using Blast2GO software resulted in information for 60% of the unigenes in the assembly. The second transcriptome assembly was constructed from more than 200 million Illumina Genome Analyzer II reads (80-120 nt) using a combination of Velvet, CLC workbench and CAP3 software packages. BWA, SAMtools and in-house Perl scripts were used to identify SNPs among three pepper genotypes. The SNPs were filtered to be at least 50 bp from any intron-exon junctions as well as flanking SNPs. More than 22,000 high-quality putative SNPs were identified. Using the MISA software, 10,398 SSR markers were also identified within the Illumina transcriptome assembly and primers were designed for the identified markers. The assembly was annotated by Blast2GO and 14,740 (12%) of annotated contigs were associated with functional proteins.

CONCLUSIONS

Before availability of pepper genome sequence, assembling transcriptomes of this economically important crop was required to generate thousands of high-quality molecular markers that could be used in breeding programs. In order to have a better understanding of the assembled sequences and to identify candidate genes underlying QTLs, we annotated the contigs of Sanger-EST and Illumina transcriptome assemblies. These and other information have been curated in a database that we have dedicated for pepper project.

Complementary DNAs encoding rat kidney chloride channels (ClC-K2L and ClC-K2S) were isolated by a polymerase chain reaction cloning strategy. Degenerate primers were designed based on the significant amino acid identity of the previously cloned chloride channels (ClC-0, -1, -2, and -K1). The 687-amino acid protein encoded by ClC-K2L is about 80% identical to rat ClC-K1 and about 40% identical to ClC-0, -1, and -2. ClC-K2S encodes a 632-amino acid protein in which 55 amino acids containing the putative second membrane-spanning domain of ClC-K2L are deleted. Chloride currents induced by both clones were very similar in terms of inhibitor sensitivity and anion selectivity (Br->> I->> Cl->>) cyclamate-). Northern blot with total ClC-K2L as a probe under high stringency revealed its message predominantly in kidney, especially in the outer and inner medulla. Reverse transcription polymerase chain reaction technique using microdissected nephron segments revealed that the main site of expression of both clones in kidney was the thick ascending limb of Henle's loop and collecting ducts, where the existence of a variety of chloride channels and their importance for maintaining body fluid homeostasis have been demonstrated. These results suggest that ClC-K2L and -K2S are chloride channels in the thick ascending limb and collecting ducts and may be important routes for transcellular chloride transport like ClC-K1.

Ion channels are frequently organized in a modular fashion and consist of a membrane-embedded pore domain and a soluble regulatory domain. A similar organization is found for the ClC family of Cl- channels and transporters. Here, we describe the crystal structure of the cytoplasmic domain of ClC-0, the voltage-dependent Cl- channel from T. marmorata. The structure contains a folded core of two tightly interacting cystathionine beta-synthetase (CBS) subdomains. The two subdomains are connected by a 96 residue mobile linker that is disordered in the crystals. As revealed by analytical ultracentrifugation, the domains form dimers, thereby most likely extending the 2-fold symmetry of the transmembrane pore. The structure provides insight into the organization of the cytoplasmic domains within the ClC family and establishes a framework for guiding future investigations on regulatory mechanisms.

Endocytosis performs a wide range of functions in animals and plants. Clathrin-coated vesicle (CCV) formation is an initial step of endocytosis, and in animal cells is largely achieved by dynamins. However, little is known of its molecular mechanisms in plant cells. To identify dynamin-related proteins (DRPs) involved in endocytic CCV formation in plant cells, we compared the behaviors of two structurally different Arabidopsis DRPs, DRP2B and DRP1A, with those of the clathrin light chain (CLC), a marker of CCVs, at the plasma membrane by variable incidence angle fluorescent microscopy (VIAFM). DRP2B shares domain organization with animal dynamins whereas DRP1A is plant-specific. We show that green fluorescent protein (GFP)-tagged DRP2B and DRP1A colocalized with CLC tagged with monomeric Kusabira Orange (mKO) in Arabidopsis cultured cells. Time-lapse VIAFM observations suggested that both GFP-DRP2B and GFP-DRP1A appeared and accumulated on the existing mKO-CLC foci and disappeared at the same time as or immediately after the disappearance of mKO-CLC. Moreover, DRP2B and DRP1A colocalized and assembled/disassembled together at the plasma membrane in Arabidopsis cells. A yeast two-hybrid assay showed that DRP2B and DRP1A interacted with each other. An inhibitor of clathrin-mediated endocytosis, tyrphostin A23, disturbed the localization of DRP1A, but had little effect on the localization of DRP2B, indicating that DRP1A and DRP2B have different molecular properties. These results suggest that DRP2B and DRP1A participate together in endocytic CCV formation in Arabidopsis cells despite the difference of their molecular properties.

It is now evident that the term Bartter syndrome does not represent a unique entity but encompasses a variety of disorders of renal electrolyte transport. Application of molecular biology techniques has permitted a better understanding of these "Bartter-like syndromes," which at present can be divided into three different genetic and clinical entities. Neonatal Bartter syndrome is observed in newborn infants and characterized by polyhydramnios, premature delivery, life-threatening episodes of fever and dehydration during the early weeks of life, growth retardation, hypercalciuria, and early-onset nephrocalcinosis. Two molecular defects have been identified: either at the gene encoding the renal bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) or the gene encoding an ATP-sensitive inwardly rectifying K channel (ROMK). "Classic" Bartter syndrome is mostly observed during infancy and childhood and is characterized clinically by polyuria and growth retardation. Nephrocalcinosis is not present. Very recently, either deletions or mutations at the gene encoding a renal chloride channel (ClC-Kb) have been identified. Gitelman syndrome is observed in older children and adults presenting with intermittent episodes of muscle weakness and tetany, hypokalemia, and hypomagnesemia. Mutations at the gene encoding the thiazide-sensitive Na-Cl cotransporter have been identified in the majority of patients studied. Obviously the validity of this classification must be confirmed in the near future when all mutations have been described and genotypic-phenotypic correlations are better defined.

Clathrin-coated vesicles (CCV) are necessary for selective transport events, including receptor-mediated endocytosis on the plasma membrane and cargo molecule sorting in the trans-Golgi network (TGN). Components involved in CCV formation include clathrin heavy and light chains and several adaptor proteins that are conserved among plants. Clathrin-dependent endocytosis has been shown to play an integral part in plant endocytosis. However, little information is known about clathrin dynamics in living plant cells. In this study, we have visualized clathrin in Arabidopsis thaliana by tagging clathrin light chain with green fluorescent protein (CLC-GFP). Quantitative evaluations of colocalization demonstrate that the majority of CLC-GFP is localized to the TGN, and a minor population is associated with multivesicular endosomes and the Golgi trans-cisternae. Live imaging further demonstrated the presence of highly dynamic clathrin-positive tubules and vesicles, which appeared to mediate interactions between the TGNs. CLC-GFP is also targeted to cell plates and the plasma membrane. Although CLC-GFP colocalizes with a dynamin isoform at the plasma membrane, these proteins exhibit distinct distributions at newly forming cell plates. This finding indicates independent functions of CLC (clathrin light chains) and dynamin during the formation of cell plates. We have also found that brefeldin A and wortmannin treatment causes distinctly different alterations in the dynamics and distribution of clathrin-coated domains at the plasma membrane. This could account for the different effects of these drugs on plant endocytosis.

ClC-3 is a member of the ClC family of anion channels/transporters. Recently, the closely related proteins ClC-4 and ClC-5 were shown to be Cl(-)/H(+) antiporters (39, 44). The function of ClC-3 has been controversial. We studied anion currents in HEK293T cells expressing wild-type or mutant ClC-3. The basic biophysical properties of ClC-3 currents were very similar to those of ClC-4 and ClC-5, and distinct from those of the swelling-activated anion channel. ClC-3 expression induced currents with time-dependent activation that rectified sharply in the outward direction. The reversal potential of the current shifted by -48.3 +/- 2.5 mV per 10-fold (decade) change in extracellular Cl(-) concentration, which did not conform to the behavior of an anion-selective channel based upon the Nernst equation, which predicts a -58.4 mV/decade shift at 22 degrees C. Manipulation of extracellular pH (6.35-8.2) altered reversal potential by 10.2 +/- 3.0 mV/decade, suggesting that ClC-3 currents were coupled to proton movement. Mutation of a specific glutamate residue (E224A) changed voltage dependence in a manner similar to that observed in other ClC Cl(-)/H(+) antiporters. Mutant currents exhibited Nernstian changes in reversal potential in response to altered extracellular Cl(-) concentration that averaged -60 +/- 3.4 mV/decade and were pH independent. Thus ClC-3 overexpression induced a pH-sensitive conductance in HEK293T cells that is biophysically similar to ClC-4 and ClC-5.

A defect in the yeast GEF1 gene, a CLC chloride channel homolog leads to an iron requirement and cation sensitivity. The iron requirement is due to a failure to load Cu2+ onto a component of the iron uptake system, Fet3. This process, which requires both Gef1 and the Menkes disease Cu2+-ATPase yeast homolog Ccc2, occurs in late- or post-Golgi vesicles, where Gef1 and Ccc2 are localized. The defects of gef1 mutants can be suppressed by the introduction of Torpedo marmorata CLC-0 or Arabidopsis thaliana CLC-c and -d chloride channel genes. The functions of Gef1 in cation homeostasis provide clues to the understanding of diseases caused by chloride channel mutations in humans and cation toxicity in plants.

BACKGROUND

Gitelman syndrome (GS) and Bartter syndrome (BS) are hereditary hypokalemic tubulopathies with distinct phenotypic features. GS has been considered a genetically homogeneous disorder caused by mutation in the gene encoding the NaCl cotransporter (TSC) of the distal convoluted tubule. In contrast, BS is caused by mutations in the genes encoding either the Na-K-2Cl cotransporter (NKCC2), the K+ channel (ROMK) or the Cl- channel (ClC-Kb) of the thick ascending limb. The purpose of this study was to examine the clinical, biochemical and genetic characteristics of a very large inbred Bedouin kindred in Northern Israel with hereditary hypokalemic tubulopathy.

METHODS

Twelve family members affected with hypokalemic tubulopathy, as well as 26 close relatives were clinically and biochemically evaluated. All study participants underwent genetic linkage analysis. Mutation analysis was performed in affected individuals.

RESULTS

Evaluation of affected family members (age range 3 to 36 years) revealed phenotypic features of both GS and classic Bartter syndrome (CBS). Features typical of GS included late age of presentation (>15 years) in 7 patients (58%), normal growth in 9 (75%), hypomagnesemia (SMg <0.7mmol/L) in 5 (42%), hypermagnesiuria (FEMg>5%) in 6 (50%) and hypocalciuria (urinary calcium/creatinine mmol/mmol <0.15) in 5 (42%). Features typical of CBS included early age of presentation (<1 year) in 3 (25%), polyuria/dehydration in 4 (33%), growth retardation in 3 (25%), hypercalciuria (urinary calcium/creatinine mmol/mmoverline>0.55) in 4 (33%) and nephrolithiasis in 1 (8%). Linkage analysis in affected patients excluded the TSC gene, SLC12A3, as the mutated gene, but demonstrated linkage to the Cl- channel gene, CLCNKB, on chromosome 1p36. Mutation analysis by direct sequencing revealed a novel homozygous missense mutation, arginine 438 to histidine (R438H), in exon 13 of CLCNKB in all patients. A restriction fragment length polymorphism (RFLP) analysis has been developed to aid in genotyping of family members.

CONCLUSIONS

Our findings demonstrate intrafamilial heterogeneity, namely the presence of GS and CBS phenotypes, in a kindred with the CLCNKB R438H mutation. We conclude that GS can be caused by a mutation in a gene other than SLC12A3. The exact role of the CLCNKB R438H mutation in the pathogenesis of the electrolyte and mineral abnormalities in GS and CBS remains to be established.