Chloride Channel Family Research Articles

Functional expression of the TMEM16 family of calcium-activated chloride channels in airway smooth muscle

Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of eight members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiological recordings, which were blocked by using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiological properties of CaCCs, and they modulate contractile tone in airway smooth muscle. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma.

Functional characterization of a ClC transporter by solid-supported membrane electrophysiology

EcClC, a prokaryotic member of the ClC family of chloride channels and transporters, works as coupled H+/Cl− exchanger. With a known structure and the possibility of investigating its behavior with different biochemical and biophysical techniques, the protein has become an important model system for the family. Although many aspects of its function have been previously characterized, it was difficult to measure transport on the same sample under different environmental conditions. To overcome this experimental limitation, we have studied EcClC by solid-supported membrane electrophysiology. The large transport-related transient currents and a simple way of relating transport rates to the measured signal have allowed a thorough investigation of ion selectivity, inhibition, and the dependence of transport on changes in ion concentration and pH. Our results confirm that the protein transports larger anions with about similar rates, whereas the smaller fluoride is not a substrate. We also show that 4,4′-diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS), a known inhibitor of other anion transport protein, irreversibly inhibits EcClC from the intracellular side. The chloride dependence shows an apparent saturation at millimolar concentrations that resembles a similar behavior in eukaryotic ClC channels. Our experiments have also allowed us to quantify the pH dependence of transport. EcClC shows a strong activation at low pH with an apparent pKa of 4.6. The pronounced pH dependence is lost by the mutation of a conserved glutamate facing the extracellular solution that was previously shown to be an acceptor for transported protons, whereas it is largely retained by the mutation of an equivalent residue at the intracellular side. Our results have provided a quantitative basis for the transport behavior of EcClC, and they will serve as a reference for future investigations of novel electrogenic transporters with still-uncharacterized properties.

Surprises from an Unusual CLC Homolog

The chloride channel (CLC) family is distinctive in that some members are Cl− ion channels and others are Cl−/H+ antiporters. The molecular mechanism that couples H+ and Cl− transport in the antiporters remains unknown. Our characterization of a novel bacterial homolog from Citrobacter koseri, CLC-ck2, has yielded surprising discoveries about the requirements for both Cl− and H+ transport in CLC proteins. First, even though CLC-ck2 lacks conserved amino acids near the Cl−-binding sites that are part of the CLC selectivity signature sequence, this protein catalyzes Cl− transport, albeit slowly. Ion selectivity in CLC-ck2 is similar to that in CLC-ec1, except that SO42− strongly competes with Cl− uptake through CLC-ck2 but has no effect on CLC-ec1. Second, and even more surprisingly, CLC-ck2 is a Cl−/H+ antiporter, even though it contains an isoleucine at the Gluin position that was previously thought to be a critical part of the H+ pathway. CLC-ck2 is the first known antiporter that contains a nonpolar residue at this position. Introduction of a glutamate at the Gluin site in CLC-ck2 does not increase H+ flux. Like other CLC antiporters, mutation of the external glutamate gate (Gluex) in CLC-ck2 prevents H+ flux. Hence, Gluex, but not Gluin, is critical for H+ permeation in CLC proteins.

Macrocyclic Lactone Anthelmintics: Spectrum of Activity and Mechanism of Action

Macrocyclic lactones (MLs), exemplified by the prototype of the class, ivermectin (IVM), are mainstays of programs for the control of nematode and arthropod parasites and pests. Since their introduction 30 years ago, research has revealed that they act on a family of ligand-gated chloride channels gated by glutamate, which is largely restricted to animals in the phyla Nematoda and Arthropoda. Studies on IVM in model organisms have contributed greatly to our understanding of ML pharmacology, but our understanding of the basis for differences among species and among MLs in potency and spectrum remains far from complete.

Endoplasmic reticulum potassium–hydrogen exchanger and small conductance calcium-activated potassium channel activities are essential for ER calcium uptake in neurons and cardiomyocytes

Calcium pumping into the endoplasmic reticulum (ER) lumen is thought to be coupled to a countertransport of protons through sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) and the members of the ClC family of chloride channels. However, pH in the ER lumen remains neutral, which suggests a mechanism responsible for proton re-entry. We studied whether cation-proton exchangers could act as routes for such a re-entry. ER Ca(2+) uptake was measured in permeabilized immortalized hypothalamic neurons, primary rat cortical neurons and mouse cardiac fibers. Replacement of K(+) in the uptake solution with Na(+) or tetraethylammonium led to a strong inhibition of Ca(2+) uptake in neurons and cardiomyocytes. Furthermore, inhibitors of the potassium-proton exchanger (quinine or propranolol) but not of the sodium-proton exchanger reduced ER Ca(2+) uptake by 56-82%. Externally added nigericin, a potassium-proton exchanger, attenuated the inhibitory effect of propranolol. Inhibitors of small conductance calcium-sensitive K(+) (SK(Ca)) channels (UCL 1684, dequalinium) blocked the uptake of Ca(2+) by the ER in all preparations by 48-94%, whereas inhibitors of other K(+) channels (IK(Ca), BK(Ca) and K(ATP)) had no effect. Fluorescence microscopy and western blot analysis revealed the presence of both SK(Ca) channels and the potassium-proton exchanger leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1) in ER in situ and in the purified ER fraction. The data obtained demonstrate that SK(Ca) channels and LETM1 reside in the ER membrane and that their activity is essential for ER Ca(2+) uptake.

ClC-2 contributes to tonic inhibition mediated by α5 subunit-containing GABAA receptor in experimental temporal lobe epilepsy

Temporal lobe epilepsy (TLE), characterized by spontaneous recurrent seizures, learning and memory impairments is associated with neurodegeneration, abnormal reorganization of the circuitry and loss of functional inhibition in hippocampus. In adult hippocampus, the GABAergic cells mediate the major inhibitory function of the principal neurons, promoting the Cl(-) entry through the GABA(A) receptor, whether through phasic (synaptic) or tonic (extrasynaptic) conductance. Aside from classical synaptic component, tonic GABAergic inhibition mediated by extrasynaptic GABA(A) receptor received increasing attention over the past years. There is growing evidence that tonic inhibition plays an important role in epilepsy, memory and cognition. Since GABA(A) receptor-mediated inhibition depends on the maintenance of intracellular Cl(-) concentration at low levels in mature neurons, a shift in E(cl) is likely to participate in the generation and not merely a consequence of TLE. As we know, chloride channel-2 (ClC-2) is a member of the supergene family of voltage-gated chloride channels, it constitutes part of the background conductance and is involved in chloride extrusion. Here we show that ClC-2 were upregulated functionally in CA1 pyramidal cells in pilocarpine-treated rats, and that an observed increase in ClC-2 currents in CA1 pyramidal cells was reversed by L655,708, a specific antagonist of α5 subunit-containing GABA(A) receptor.

Abstract 876: Genetic variation in the voltage-gated chloride channel family members CLCN2 and CLCN3 in relation to glioma risk and outcome

Abstract Gliomas account for the majority of primary brain neoplasias and are among the most lethal human tumors. Tumor cells diffusely infiltrate surrounding brain tissue. The invasion of glioma cells into normal brain is thought to be facilitated by ion channels which dynamically regulate cell volume and the ability of tumor cells to shrink and invade through tortuous extracellular spaces in the brain. To shrink, glioma cells release K+ and Cl- through ion channels, which induce water to leave the cells through aquaporins. Recent evidence suggests that CL- transport in glioma cells is mediated primarily by the voltage-gated chloride channel family members, CLCN2 and CLCN3, making them attractive candidate genes in glioma pathogenesis. In the present study, we tested whether single nucleotide polymorphisms (SNPs) in CLCN2 (3q27) and CLCN3 (4q33) are associated with glioma risk or patient outcome in a clinic-based case-control study conducted at medical centers in the southeastern US. A total of 15 candidate and haplotype tagging SNPs (4 SNPs in CLCN2 and 11 SNPs in CLCN3) were genotyped using the Illumina Goldengate assay in 563 newly diagnosed (eg. nonrecurrent) glioma cases (including 324 WHO grade IV glioblastomas (GBM); 145 WHO grade II or III astrocytomas and 94 oligoastrocytomas and oligodendrogliomas) and 629 healthy controls. DNA was isolated from saliva samples. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) for glioma risk in relation to individual genotypes adjusting for age and gender. Proportional hazards regression was used to estimate age and gender-adjusted hazard ratios (HR) for GBM-related death (203 deaths; median follow up: 11.2 months) for each SNP. No consistent associations were observed for any examined variant in CLCN2. In contrast, 6 of the examined SNPs in CLCN3, all in moderate linkage disequilibrium (r2: 0.30-0.80), were significantly associated with glioma risk. Among them, the strongest signal was observed in rs3797040 (NT_016354.19:g.95088578C>T), a SNP located in a putative transcription binding site (per variant “T” allele OR: 1.25; 95% CI:1.05-1.47; p=0.0095; allele frequency: 0.49); the observed excess risk associated with the variant rs3797040 allele was consistent in GBM (per allele OR: 1.23; 95% CI: 1.01-1.51; p=0.042), astrocytomas (per allele OR: 1.15; 95% CI: 0.88-1.49; p=0.312) and oligodendrogliomas (per allele OR: 1.75; 95% CI: 1.23-2.44; p=0.001). The ancestral “C” allele in CLCN3 rs3797040 was associated with increased mortality rates among the patients with GBM (per allele HR: 1.31; 95% CI: 1.06-1.62; p=0.011). To our knowledge this is the first report suggesting that genetic variation in plasma membrane ion channels may be a determinant of glioma risk. Further studies are needed to confirm these observations and identify the putative causal variant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 876. doi:10.1158/1538-7445.AM2011-876

Expression and Localization of CLC Chloride Transport Proteins in the Avian Retina

Members of the ubiquitously expressed CLC protein family of chloride channels and transporters play important roles in regulating cellular chloride and pH. The CLCs that function as Cl−/H+ antiporters, ClCs 3–7, are essential in particular for the acidification of endosomal compartments and protein degradation. These proteins are broadly expressed in the nervous system, and mutations that disrupt their expression are responsible for several human genetic diseases. Furthermore, knock-out of ClC3 and ClC7 in the mouse result in the degeneration of the hippocampus and the retina. Despite this evidence of their importance in retinal function, the expression patterns of different CLC transporters in different retinal cell types are as yet undescribed. Previous work in our lab has shown that in chicken amacrine cells, internal Cl− can be dynamic. To determine whether CLCs have the potential to participate, we used PCR and immunohistochemical techniques to examine CLC transporter expression in the chicken retina. We observed a high level of variation in the retinal expression levels and patterns among the different CLC proteins examined. These findings, which represent the first systematic investigation of CLC transporter expression in the retina, support diverse functions for the different CLCs in this tissue.

The Tao of Chloride Transporter Structure

A protein structure from a heat-loving alga offers insight into the diverse family of CLC chloride channels and transporters.

The Arabidopsis vacuolar anion transporter, AtCLCc, is involved in the regulation of stomatal movements and contributes to salt tolerance

In plant cells, anion channels and transporters are essential for key functions such as nutrition, resistance to biotic or abiotic stresses, and ion homeostasis. In Arabidopsis, members of the chloride channel (CLC) family located in intracellular organelles have been shown to be required for nitrate homeostasis or pH adjustment, and previous results indicated that AtCLCc is involved in nitrate accumulation. We investigated new physiological functions of this CLC member in Arabidopsis. Here we report that AtCLCc is strongly expressed in guard cells and pollen and more weakly in roots. Use of an AtCLCc:GFP fusion revealed localization to the tonoplast. Disruption of the AtCLCc gene by a T-DNA insertion in four independent lines affected physiological responses that are directly related to the movement of chloride across the tonoplast membrane. Opening of clcc stomata was reduced in response to light, and ABA treatment failed to induce their closure, whereas application of KNO₃ but not KCl restored stomatal opening. clcc mutant plants were hypersensitive to NaCl treatment when grown on soil, and to NaCl and KCl in vitro, confirming the chloride dependence of the phenotype. These phenotypes were associated with modifications of chloride content in both guard cells and roots. These data demonstrate that AtCLCc is essential for stomatal movement and salt tolerance by regulating chloride homeostasis.

CLCA2, a novel RUNX1 partner gene in a therapy-related leukemia with t(1;21)(p22;q22)

The RUNX1 gene is frequently rearranged in de novo and therapy-related leukemia. In the present study, we identified the CLCA2 gene as a novel fusion partner of RUNX1 in a case of therapy-related acute myeloid leukemia associated with t(1;21)(p22;q22). Reverse transcriptase–polymerase chain reaction analysis and sequencing revealed that the t(1;21) results in out-of-frame RUNX1-CLCA2 fusions. Alternative splicing generates at least six fusion transcripts, including a major transcript fusing RUNX1 exon 6 with CLCA2 exon 2. These out-of-frame fusions produce putative truncated RUNX1 isoforms retaining the DNA binding Runt domain but not the transcriptional regulatory domain of RUNX1. No mutations were found in the exons encoding the Runt and C-terminal domains of the nonrearranged RUNX1 gene. Similar to truncated RUNX1 isoforms previously described, these shortened products could act as dominant negative inhibitors of RUNX1-dependent transactivation. CLCA2 is a breast tumor suppressor gene that encodes a member of the calcium-activated chloride channel family and is involved for the first time in a chromosomal translocation. The RUNX1-CLCA2 fusion is another example of out-of-frame fusion generating truncated RUNX1 isoforms that represent a recurrent molecular mechanism in RUNX1-related leukemias.

The proline 160 in the selectivity filter of the Arabidopsis NO3−/H+ exchanger AtCLCa is essential for nitrate accumulation in planta

Nitrate, the major nitrogen source for plants, can be accumulated in the vacuole. Its transport across the vacuolar membrane is mediated by AtCLCa, an antiporter of the chloride channel (CLC) protein family. In contrast to other CLC family members, AtCLCa transports nitrate coupled to protons. Recently, the different behaviour towards nitrate of CLC proteins has been linked to the presence of a serine or proline in the selectivity filter motif GXGIP. By monitoring AtCLCa activity in its native environment, we show that if proline 160 in AtCLCa is changed to a serine (AtCLCa(P160S) ), the transporter loses its nitrate selectivity, but the anion proton exchange mechanism is unaffected. We also performed in vivo analyses in yeast and Arabidopsis. In contrast to native AtCLCa, expression of AtCLCa(P160S) does not complement either the ΔScCLC yeast mutant grown on nitrate or the nitrate under-accumulation phenotype of clca knockout plants. Our results confirm the significance of this amino acid in the conserved selectivity filter of CLC proteins and highlight the importance of the proline in AtCLCa for nitrate metabolism in Arabidopsis.

The LysR-Type Virulence Activator AphB Regulates the Expression of Genes in Vibrio cholerae in Response to Low pH and Anaerobiosis

AphB is a LysR-type activator that initiates the expression of the virulence cascade in Vibrio cholerae by cooperating with the quorum-sensing-regulated activator AphA at the tcpPH promoter on the Vibrio pathogenicity island (VPI). To identify the ancestral chromosomal genes in V. cholerae regulated by AphB, we carried out a microarray analysis and show here that AphB influences the expression of a number of genes that are not associated with the VPI. One gene strongly activated by AphB is cadC, which encodes the ToxR-like transcriptional activator responsible for activating the expression of lysine decarboxylase, which plays an important role in survival at low pH. Other genes activated by AphB encode a Na(+)/H(+) antiporter, a carbonic anhydrase, a member of the ClC family of chloride channels, and a member of the Gpr1/Fun34/YaaH family. AphB influences each of these genes directly by recognizing a conserved binding site within their promoters, as determined by gel mobility shift assays. Transcriptional lacZ fusions indicate that AphB activates the expression of these genes under aerobic conditions in response to low pH and also under anaerobic conditions at neutral pH. Further experiments show that the regulation of cadC by AphB in response to low pH and anaerobiosis is mirrored in the heterologous organism Escherichia coli, is independent of the global regulators Fnr and ArcAB, and depends upon the region of the promoter that contains the AphB binding site. These results raise the possibility that the activity of AphB is influenced by the pH and oxygen tension of the environment.

Expression patterns of ClC-3 mRNA and protein in aortic smooth muscle, kidney and brain in diabetic rats

ClC-3, a member of the ClC family of voltage-gated chloride channels, regulates cell proliferation of cultured rat aortic vascular smooth muscle cells, pathogenesis of allergic rhinitis and tumor cell migration. However, its role in diabetic animals is still unknown. To address this issue, we investigated the expression patterns of ClC-3 in diabetic rats. Five-week-old Sprague-Dawley rats were divided into two groups, 50 non-diabetic control rats (non-DM) and 50 diabetic model rats (DM). ClC-3 mRNA and protein expression in aortic smooth muscle, kidney and brain tissues were examined by fluorimeter-based quantitive RT-PCR assay and Western blot analysis, respectively. ClC-3 mRNA and protein were endogenously expressed in aortic smooth muscle, kidney (cortex and medulla) and brain tissues of both control and streptozotocin-induced diabetic rats. ClC-3 mRNA and protein expression levels were significantly higher in aortic smooth muscle and brain tissues of diabetic rats, but significantly decreased in kidney medulla tissue, relative to non-DM controls. There were no significant differences in ClC-3 mRNA and protein expression in kidney cortex between non-diabetic control and diabetic rats. Furthermore, the altered ClC-3 expression patterns in diabetic rat aortic smooth muscle, brain, and kidney medulla tissues all correlated with the changes in blood glucose levels (p < 0.05). In conclusion, our data show for the first time that diabetes alters both the gene and protein expression of ClC-3 channels. These changes may contribute to the impaired vascular, brain and kidney functions observed in diabetes.

Chloride channel ClC-3 in gills of the euryhaline teleost,Tetraodon nigroviridis: expression, localization and the possible role of chloride absorption

Previous studies have reported the mechanisms of ion absorption and secretion by diverse membrane transport proteins in gills of various teleostean species. To date, however, the chloride channel expressed in the basolateral membrane of mitochondrion-rich (MR) cells for Cl(-) uptake in freshwater (FW) fish is still unknown. In this study, the combination of bioinformatics tools [i.e. National Center for Biotechnology Information (NCBI) database, Tetraodon nigroviridis (spotted green pufferfish) genome database (Genoscope), BLAT and BLASTn] were used to identify the gene of ClC-3 (TnClC-3), a member of the CLC chloride channel family in the T. nigroviridis genome. RT-PCR analysis revealed that the gene encoding for the ClC-3 protein was widely expressed in diverse tissues (i.e. gill, kidney, intestine, liver and brain) of FW- and seawater (SW)-acclimated pufferfish. In whole-mount double immunofluorescent staining, branchial ClC-3-like immunoreactive protein was localized to the basolateral membrane of Na(+)/K(+)-ATPase (NKA) immunoreactive cells in both the FW- and SW-acclimated pufferfish. In response to salinity, the levels of transcript of branchial TnClC-3 were similar between FW and SW fish. Moreover, the membrane fraction of ClC-3-like protein in gills was 2.7-fold higher in FW compared with SW pufferfish. To identify whether the expression of branchial ClC-3-like protein specifically responded to lower environmental [Cl(-)], the pufferfish were acclimated to artificial waters either with a normal (control) or lower Cl(-) concentration (low-Cl). Immunoblotting of membrane fractions of gill ClC-3-like protein showed the expression was about 4.3-fold higher in pufferfish acclimated to the low-Cl environment than in the control group. Furthermore, branchial ClC-3-like protein was rapidly elevated in response to acute changes of environmental salinity or [Cl(-)]. Taken together, pufferfish ClC-3-like protein was expressed in the basolateral membrane of gill MR cells, and the protein amounts were stimulated by hyposmotic and low-Cl environments. The enhancement of ClC-3-like protein may trigger the step of basolateral Cl(-) absorption of the epithelium to carry out iono- and osmoregulatory functions of euryhaline pufferfish gills.

ClC transporters: discoveries and challenges in defining the mechanisms underlying function and regulation of ClC-5

The involvement of several members of the chloride channel (ClC) family of membrane proteins in human disease highlights the need to define the mechanisms underlying their function and the consequences of disease-causing mutations. Despite the utility of high-resolution structural models, our understanding of the molecular basis for function of the chloride channels and transporters in the family remains incomplete. In this review, we focus on recent discoveries regarding molecular mechanisms underlying the regulated chloride:proton antiporter activity of ClC-5, the protein mutated in the Dent's disease-a kidney disease presenting with proteinuria and renal failure in severe cases. We discuss the putative role of ClC-5 in receptor-mediated endocytosis and protein uptake by the proximal renal tubule and the possible molecular and cellular consequences of disease-causing mutations. However, validation of these models will require future study of the intrinsic function of this transporter, in situ, in the membranes of recycling endosomes in proximal tubule epithelial cells.

Expression of Genes Encoding Multi-Transmembrane Proteins in Specific Primate Taste Cell Populations

BackgroundUsing fungiform (FG) and circumvallate (CV) taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed.Methodology/Principal FindingsUsing double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive), sour cells (PKD2L1-positive), as well as other taste cell populations. Transmembrane protein 44 (TMEM44), a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1), a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1), a calcium-binding transmembrane protein; and anoctamin 7 (ANO7), a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B), a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release.Conclusions/SignificanceIdentification of genes encoding multi-transmembrane domain proteins expressed in primate taste buds provides new insights into the processes of taste cell development, signal transduction, and information coding. Discrete taste cell populations exhibit highly specific gene expression patterns, supporting a model whereby each mature taste receptor cell is responsible for sensing, transmitting, and coding a specific taste quality.

ClC-5 regulates dentin development through TGF-β1 pathway

ClC-5 is one of the voltage-dependent chloride channel (ClC) family members. Mutations involving CLCN5 cause an X-linked nephropathy associated with Dent's disease. Some Clcn5 gene knockout (ClC-5 KO) mice have abnormal growth of the teeth; however, the expression and function of ClC-5 during tooth development is still unknown. Herein we report abnormal dentin structure, decreased DSPP and increased TGF-β1 protein level in ClC-5 KO teeth. In odontoblast-like MDPC-23 cells, the mRNA levels of Tgfb1, Dspp and Dmp-1 were upregulated with Clcn5 RNAi after 48 h treatment; whilst there was no change in those of TGF-beta receptor Tgfbr1 and Tgfbr2. We suggest that the dentin changes in ClC-5 KO mice might be a result of increasing TGF-β1, and the interplay between ClC-5 and TGF-β1 needs further identified.

The ligand-gated chloride channel gene family of Drosophila melanogaster

The antagonism of cellular excitability in insects is mediated by a family of ionotropic receptors, the ligand-gated chloride channels. In insects these inhibitory receptors include synaptic GABA and histamine receptors, functionally analogous to vertebrate GABA and glycine receptors, and glutamate receptors that appear to be unique to invertebrates. The ligand-gated chloride channel family in insects includes two well-validated targets for insect control agents: GABA receptors, which are the targets for a structural variety of small-molecule insecticides (polychlorocycloalkanes such as dieldrin and phenylpyrazoles such as fipronil) that have been used widely in agriculture; and glutamate receptors, which are the targets of macrocyclic lactone natural products (the avermectins) that have yielded not only commercial insecticides but also anthelminthic agents (i.e., ivermectin) employed in animal and human health. The availability of the complete genome sequence of the insect model system Drosophila melanogaster has permitted the identification of all of the genes encoding proteins with structural similarity to known ligand-gated chloride channel subunits (ligand-gated chloride channel homologs or LCCHs). Here, we review the present status of knowledge of the structure and function of the proteins encoded by this compact gene family. The 12 LCCH genes of D. melanogaster exhibit a surprising degree of structural diversity, which is further enhanced for some subunits by a variety of post-transcriptional and post-translational modifications. Although the structures of the gene products encoded by this small gene family are now well characterized, surprisingly little is known of the biological functions of the majority of them and the structures of most native receptors remain unknown.

ATP Binding to the C Terminus of the Arabidopsis thaliana Nitrate/Proton Antiporter, AtCLCa, Regulates Nitrate Transport into Plant Vacuoles

Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO(3)(-)/H(+) exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine beta-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of nitrate influx into the vacuole at cytosolic physiological nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.