Cellular basis of ClC-2 Cl- channel-related brain and testis pathologies.
Ontology highlight
ABSTRACT:
SUBMITTER: Goppner C
PROVIDER: S-EPMC7949093 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The CLC family of Cl(-)-transporting proteins includes both Cl(-) channels and Cl(-)/H(+) exchange transporters. CLC-ec1, a structurally known bacterial homolog of the transporter subclass, exchanges two Cl(-) ions per proton with strict, obligatory stoichiometry. Point mutations at two residues, Glu(148) and Tyr(445), are known to impair H(+) movement while preserving Cl(-) transport. In the x-ray crystal structure of CLC-ec1, these residues form putative "gates" flanking an ion-binding region. In mutants with both of the gate-forming side chains reduced in size, H(+) transport is abolished, and unitary Cl(-) transport rates are greatly increased, well above values expected for transporter mechanisms. Cl(-) transport rates increase as side-chain volume at these positions is decreased. The crystal structure of a doubly ungated mutant shows a narrow conduit traversing the entire protein transmembrane width. These characteristics suggest that Cl(-) flux through uncoupled, ungated CLC-ec1 occurs via a channel-like electrodiffusion mechanism rather than an alternating-exposure conformational cycle that has been rendered proton-independent by the gate mutations.
| S-EPMC2516207 | biostudies-literature
Project description:GlialCAM, a glial cell adhesion molecule mutated in megalencephalic leukoencephalopathy with subcortical cysts, targets the CLC-2 Cl(-) channel to cell contacts in glia and activates CLC-2 currents in vitro and in vivo. We found that GlialCAM clusters all CLC channels at cell contacts in vitro and thus studied GlialCAM interaction with CLC channels to investigate the mechanism of functional activation. GlialCAM slowed deactivation kinetics of CLC-Ka/barttin channels and increased CLC-0 currents opening the common gate and slowing its deactivation. No functional effect was seen for common gate deficient CLC-0 mutants. Similarly, GlialCAM targets the common gate deficient CLC-2 mutant E211V/H816A to cell contacts, without altering its function. Thus, GlialCAM is able to interact with all CLC channels tested, targeting them to cell junctions and activating them by stabilizing the open configuration of the common gate. These results are important to better understand the physiological role of GlialCAM/CLC-2 interaction.
| S-EPMC4156679 | biostudies-literature
Project description:Ion channels have historically been viewed as distinct from secondary active transporters. However, the recent discovery that the CLC 'chloride channel' family is made up of both channels and active transporters has led to the hypothesis that the ion-transport mechanisms of these two types of membrane proteins may be similar. Here we use single-channel analysis to demonstrate that ClC-0 channel gating (opening and closing) involves the transmembrane movement of protons. This result indicates that ClC-0 is a 'broken' Cl(-)/H(+) antiporter in which one of the conformational states has become leaky for chloride ions. This finding clarifies the evolutionary relationship between the channels and transporters and conveys that similar mechanisms and analogous protein movements are used by both.
| S-EPMC2559860 | biostudies-literature
Project description:The renal collecting duct plays a critical role in setting urinary volume and composition, with principal cells transporting Na+ and K+ and intercalated cells mediating Cl- reabsorption. Published evidence implies Angiotensin II (Ang II) is a potent regulator of the collecting duct apical transport systems in response to systemic volume depletion. However, virtually nothing is known about Ang II actions on the basolateral conductance of principal and intercalated cells. Here, we combined macroscopic and single channel patch clamp recordings from freshly isolated mouse collecting ducts with biochemical and fluorescence methods to demonstrate an acute stimulation of the basolateral Cl- conductance and specifically the ClC-K2 Cl- channel by nanomolar Ang II concentrations in intercalated cells. In contrast, Ang II did not exhibit measurable effects on the basolateral conductance and on Kir4.1/5.1 potassium channel activity in principal cells. Although both Ang II receptors AT1 and AT2 are expressed in collecting duct cells, we show that AT1 receptors were essential for stimulatory actions of Ang II on ClC-K2. Moreover, AT1R-/- mice had decreased renal ClC-K2 expression. We further demonstrated that activation of NADPH oxidases is the major signaling pathway downstream of Ang II-AT1R that leads to stimulation of ClC-K2. Treatment of freshly isolated collecting ducts with Ang II led to production of reactive oxygen species on the same timescale as single channel ClC-K2 activation. Overall, we propose that Ang II-dependent regulation of ClC-K2 in intercalated cells is instrumental for stimulation of Cl- reabsorption by the collecting duct, particularly during hypovolemic states.
| S-EPMC7949157 | biostudies-literature
Project description:Dent disease 1 (DD1) is caused by mutations in the CLCN5 gene encoding a voltage-gated electrogenic nCl-/H+ exchanger ClC-5. Using ion-selective microelectrodes and Xenopus oocytes, here we studied Cl-/H+ coupling properties of WT ClC-5 and four DD1-associated variants (S244L, R345W, Q629*, and T657S), along with trafficking and localization of ClC-5. WT ClC-5 had a 2Cl-/H+ exchange ratio at a Vh of +40 mV with a [Cl-]out of 104 mm, but the transport direction did not reverse with a [Cl-]out of 5 mm, indicating that ClC-5-mediated exchange of two Cl- out for one H+ in is not permissible. We hypothesized that ClC-5 and H+-ATPase are functionally coupled during H+-ATPase-mediated endosomal acidification, crucial for ClC-5 activation by depolarizing endosomes. ClC-5 transport that provides three net negative charges appeared self-inhibitory because of ClC-5's voltage-gated properties, but shunt conductance facilitated further H+-ATPase-mediated endosomal acidification. Thus, an on-and-off "burst" of ClC-5 activity was crucial for preventing Cl- exit from endosomes. The subcellular distribution of the ClC-5:S244L variant was comparable with that of WT ClC-5, but the variant had a much slower Cl- and H+ transport and displayed an altered stoichiometry of 1.6:1. The ClC-5:R345W variant exhibited slightly higher Cl-/H+ transport than ClC-5:S244L, but co-localized with early endosomes, suggesting decreased ClC-5:R345W membrane trafficking is perhaps in a fully functional form. The truncated ClC-5:Q629* variant displayed the lowest Cl-/H+ exchange and was retained in the endoplasmic reticulum and cis-Golgi, but not in early endosomes, suggesting the nonsense mutation affects ClC-5 maturation and trafficking.
| S-EPMC7008381 | biostudies-literature
Project description:Dent disease 1 (DD1) is a renal salt-wasting tubulopathy associated with mutations in the Cl-/H+ antiporter ClC-5. The disease typically manifests with proteinuria, hypercalciuria, nephrocalcinosis, and nephrolithiasis but is characterized by large phenotypic variability of no clear origin. Several DD1 cases have been reported lately with additional atypical hypokalemic metabolic alkalosis and hyperaldosteronism, symptoms usually associated with another renal disease termed Bartter syndrome (BS). Expression of the Bartter-like DD1 mutant ClC-5 G261E in HEK293T cells showed that it is retained in the ER and lacks the complex glycosylation typical for ClC-5 WT. Accordingly, the mutant abolished CLC ionic transport. Such phenotype is not unusual and is often observed also in DD1 ClC-5 mutants not associated with Bartter like phenotype. We noticed, therefore, that one type of BS is associated with mutations in the protein barttin that serves as an accessory subunit regulating the function and subcellular localization of ClC-K channels. The overlapping symptomatology of DD1 and BS, together with the homology between the proteins of the CLC family, led us to investigate whether barttin might also regulate ClC-5 transport. In HEK293T cells, we found that barttin cotransfection impairs the complex glycosylation and arrests ClC-5 in the endoplasmic reticulum. As barttin and ClC-5 are both expressed in the thin and thick ascending limbs of the Henle's loop and the collecting duct, interactions between the two proteins could potentially contribute to the phenotypic variability of DD1. Pathologic barttin mutants differentially regulated trafficking and processing of ClC-5, suggesting that the interaction between the two proteins might be relevant also for the pathophysiology of BS. Our findings show that barttin regulates the subcellular localization not only of kidney ClC-K channels but also of the ClC-5 transporter, and suggest that ClC-5 might potentially play a role not only in kidney proximal tubules but also in tubular kidney segments expressing barttin. In addition, they demonstrate that the spectrum of clinical, genetic and molecular pathophysiology investigation of DD1 should be extended.
| S-EPMC6206076 | biostudies-literature
Project description:Background and purposeCerebrovascular remodelling is one of the important risk factors of stroke. The underlying mechanisms are unclear. Integrin β3 and volume-regulated ClC-3 Cl(-) channels have recently been implicated as important contributors to vascular cell proliferation. Therefore, we investigated the role of integrin β3 in cerebrovascular remodelling and related Cl(-) signalling pathway.Experimental approachCl(-) currents were recorded using a patch clamp technique. The expression of integrin β3 in hypertensive animals was examined by Western blot and immunohistochemisty. Immunoprecipitation, cDNA and siRNA transfection were employed to investigate the integrin β3/Src/ClC-3 signalling.Key resultsIntegrin β3 expression was up-regulated in stroke-prone spontaneously hypertensive rats, 2-kidney 2-clip hypertensive rats and angiotensin II-infused hypertensive mice. Integrin β3 expression was positively correlated with medial cross-sectional area and ClC-3 expression in the basilar artery of 2-kidney 2-clip hypertensive rats. Knockdown of integrin β3 inhibited the proliferation of rat basilar vascular smooth muscle cells induced by angiotensin II. Co-immunoprecipitation and immunofluorescence experiments revealed a physical interaction between integrin β3, Src and ClC-3 protein. The integrin β3/Src/ClC-3 signalling pathway was shown to be involved in the activation of volume-regulated chloride channels induced by both hypo-osmotic stress and angiotensin II. Tyrosine 284 within a concensus Src phosphorylation site was the key point for ClC-3 channel activation. ClC-3 knockout significantly attenuated angiotensin II-induced cerebrovascular remodelling.Conclusions and implicationsIntegrin β3 mediates cerebrovascular remodelling during hypertension via Src/ClC-3 signalling pathway.
| S-EPMC4080971 | biostudies-literature
Project description:Synthetic transmembrane ion transport systems are emerging as new tools for anticancer therapy. Here, a series of 2-hydroxy-N 1,N 3-diarylisophthalamide-based fluorescent ion channel-forming compounds are reported. Ion transport studies across large unilamellar vesicles confirmed that the compound with two 3,5-bis(trifluoromethyl)phenyl arms is the most efficient transporter among the series and it facilitates M+/Cl- symport. The compound formed supramolecular ion channels with a single-channel conductance of 100 ± 2 pS, a diameter of 5.06 ± 0.16 Å and a permeability ratio, P Cl- /P K+ , of 8.29 ± 1. The molecular dynamics simulations of the proposed M2.11 channel (i.e. 11 coaxial layers of a dimeric rosette) with K+ and Cl- in the preequilibrated POPC lipid bilayer with water molecules illustrated various aspects of channel formation and ion permeation. Cell viability assay with the designed compounds indicated that cell death is being induced by the individual compounds which follow the order of their ion transport activity and chloride and cations play roles in cell death. The inherent fluorescence of the most active transporter was helpful to monitor its permeation in cells by confocal microscopy. The apoptosis-inducing activity upon perturbation of intracellular ionic homeostasis was established by monitoring mitochondrial membrane depolarization, generation of reactive oxygen species, cytochrome c release, activation of the caspase 9 pathway, and finally the uptake of the propidium iodide dye in the treated MCF7 cells.
| S-EPMC8157539 | biostudies-literature
Project description:Dysfunction of the endolysosomal system is often associated with neurodegenerative disease because postmitotic neurons are particularly reliant on the elimination of intracellular aggregates. Adequate function of endosomes and lysosomes requires finely tuned luminal ion homeostasis and transmembrane ion fluxes. Endolysosomal CLC Cl-/H+ exchangers function as electric shunts for proton pumping and in luminal Cl- accumulation. We now report three unrelated children with severe neurodegenerative disease, who carry the same de novo c.1658A>G (p.Tyr553Cys) mutation in CLCN6, encoding the late endosomal Cl-/H+-exchanger ClC-6. Whereas Clcn6-/- mice have only mild neuronal lysosomal storage abnormalities, the affected individuals displayed severe developmental delay with pronounced generalized hypotonia, respiratory insufficiency, and variable neurodegeneration and diffusion restriction in cerebral peduncles, midbrain, and/or brainstem in MRI scans. The p.Tyr553Cys amino acid substitution strongly slowed ClC-6 gating and increased current amplitudes, particularly at the acidic pH of late endosomes. Transfection of ClC-6Tyr553Cys, but not ClC-6WT, generated giant LAMP1-positive vacuoles that were poorly acidified. Their generation strictly required ClC-6 ion transport, as shown by transport-deficient double mutants, and depended on Cl-/H+ exchange, as revealed by combination with the uncoupling p.Glu200Ala substitution. Transfection of either ClC-6Tyr553Cys/Glu200Ala or ClC-6Glu200Ala generated slightly enlarged vesicles, suggesting that p.Glu200Ala, previously associated with infantile spasms and microcephaly, is also pathogenic. Bafilomycin treatment abrogated vacuole generation, indicating that H+-driven Cl- accumulation osmotically drives vesicle enlargement. Our work establishes mutations in CLCN6 associated with neurological diseases, whose spectrum of clinical features depends on the differential impact of the allele on ClC-6 function.
| S-EPMC7820737 | biostudies-literature
Project description:Cl–/H+ transporters of the CLC superfamily form a ubiquitous class of membrane proteins that catalyze stoichiometrically coupled exchange of Cl– and H+ across biological membranes. CLC transporters exchange H+ for halides and certain polyatomic anions, but exclude cations, F–, and larger physiological anions, such as PO43– and SO42–. Despite comparable transport rates of different anions, the H+ coupling in CLC transporters varies significantly depending on the chemical nature of the transported anion. Although the molecular mechanism of exchange remains unknown, studies on bacterial ClC-ec1 transporter revealed that Cl– binding to the central anion-binding site (Scen) is crucial for the anion-coupled H+ transport. Here, we show that Cl–, F–, NO3–, and SCN– display distinct binding coordinations at the Scen site and are hydrated in different manners. Consistent with the observation of differential bindings, ClC-ec1 exhibits markedly variable ability to support the formation of the transient water wires, which are necessary to support the connection of the two H+ transfer sites (Gluin and Gluex), in the presence of different anions. While continuous water wires are frequently observed in the presence of physiologically transported Cl–, binding of F– or NO3– leads to the formation of pseudo-water-wires that are substantially different from the wires formed with Cl–. Binding of SCN–, however, eliminates the water wires altogether. These findings provide structural details of anion binding in ClC-ec1 and reveal a putative atomic-level mechanism for the decoupling of H+ transport to the transport of anions other than Cl–.
| S-EPMC4898471 | biostudies-literature