Uncoupling endosomal CLC chloride/proton exchange causes severe neurodegeneration
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ABSTRACT:
SUBMITTER: Prof. Thomas, J. Jentsch
PROVIDER: S-SCDT-EMBOJ-2019-103358 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:CLC chloride/proton exchangers may support acidification of endolysosomes and raise their luminal Cl- concentration. Disruption of endosomal ClC-3 causes severe neurodegeneration. To assess the importance of ClC-3 Cl- /H+ exchange, we now generate Clcn3unc/unc mice in which ClC-3 is converted into a Cl- channel. Unlike Clcn3-/- mice, Clcn3unc/unc mice appear normal owing to compensation by ClC-4 with which ClC-3 forms heteromers. ClC-4 protein levels are strongly reduced in Clcn3-/- , but not in Clcn3unc/unc mice because ClC-3unc binds and stabilizes ClC-4 like wild-type ClC-3. Although mice lacking ClC-4 appear healthy, its absence in Clcn3unc/unc /Clcn4-/- mice entails even stronger neurodegeneration than observed in Clcn3-/- mice. A fraction of ClC-3 is found on synaptic vesicles, but miniature postsynaptic currents and synaptic vesicle acidification are not affected in Clcn3unc/unc or Clcn3-/- mice before neurodegeneration sets in. Both, Cl- /H+ -exchange activity and the stabilizing effect on ClC-4, are central to the biological function of ClC-3.
| S-EPMC7196918 | biostudies-literature
Project description:The regulation of luminal ion concentrations is critical for the function of, and transport between intracellular organelles. The importance of the acidic pH in the compartments of the endosomal-lysosomal pathway has been well-known for decades. Besides the V-ATPase, which pumps protons into their lumen, a variety of ion transporters and channels is involved in the regulation of the organelles' complex ion homeostasis. Amongst these are the intracellular members of the CLC family, ClC-3 through ClC-7. They localize to distinct but overlapping compartments of the endosomal-lysosomal pathway, partially with tissue-specific expression. Functioning as 2Cl-/H+ exchangers, they can support the vesicular acidification and accumulate luminal Cl-. Mutations in the encoding genes in patients and mouse models underlie severe phenotypes including kidney stones with CLCN5 and osteopetrosis or hypopigmentation with CLCN7. Dysfunction of those intracellular CLCs that are expressed in neurons lead to neuronal defects. Loss of endosomal ClC-3, which heteromerizes with ClC-4, results in neurodegeneration. Mutations in ClC-4 are associated with epileptic encephalopathy and intellectual disability. Mice lacking the late endosomal ClC-6 develop a lysosomal storage disease with reduced pain sensitivity. Human gene variants have been associated with epilepsy, and a gain-of-function mutation causes early-onset neurodegeneration. Dysfunction of the lysosomal ClC-7 leads to a lysosomal storage disease and neurodegeneration in mice and humans. Reduced luminal chloride, as well as altered calcium regulation, has been associated with lysosomal storage diseases in general. This review discusses the properties of endosomal and lysosomal Cl-/H+ exchange by CLCs and how various alterations of ion transport by CLCs impact organellar ion homeostasis and function in neurodegenerative disorders.
| S-EPMC7940362 | biostudies-literature
Project description:Members of the CLC protein family of Cl(-) channels and transporters display the remarkable ability to function as either chloride channels or Cl(-)/H(+) antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas recent data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here, we report that fusing the green fluorescent protein (GFP) to the N terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl(-)/H(+) exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. As observed in other CLC exchangers, ClC-6-mediated H(+) transport was abolished by mutations in either the "gating" or "proton" glutamate. Overexpression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl(-) > I(-) conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the "anion-coordinating" tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO(3)(-) conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl(-)/H(+) antiporter.
| S-EPMC2898453 | biostudies-literature
Project description:The CLC protein family contains plasma membrane chloride channels and the intracellular chloride-proton exchangers ClC-3-7. The latter proteins mainly reside on the various compartments of the endosomal-lysosomal system where they are involved in the luminal acidification or chloride accumulation. Although their partially overlapping subcellular distribution has been studied extensively, little is known about their targeting mechanism. In a comprehensive study we now performed pulldown experiments to systematically map the differential binding of adaptor proteins of the endosomal sorting machinery (adaptor proteins and GGAs (Golgi-localized, γ-ear containing, Arf binding)) as well as clathrin to the cytosolic regions of the intracellular CLCs. The resulting interaction pattern fitted well to the known subcellular localizations of the CLCs. By mutating potential sorting motifs, we could locate almost all binding sites, including one already known for ClC-3 and several new motifs for ClC-5, -6, and -7. The impact of the identified binding sites on the subcellular localization of CLC transporters was determined by heterologous expression of mutants. Surprisingly, some vesicular CLCs retained their localization after disruption of interaction sites. However, ClC-7 could be partially shifted from lysosomes to the plasma membrane by combined mutation of N-terminal sorting motifs. The localization of its β-subunit, Ostm1, was determined by that of ClC-7. Ostm1 was not capable of redirecting ClC-7 to lysosomes.
| S-EPMC2966069 | biostudies-literature
Project description:ClC-4 is a secondary active transporter that exchanges Cl(-) ions and H(+) with a 2:1 stoichiometry. In external SCN(-), ClC-4 becomes uncoupled and transports anions with high unitary transport rate. Upon voltage steps, the number of active transporters varies in a time-dependent manner, resembling voltage-dependent gating of ion channels. We here investigated modification of the voltage dependence of uncoupled ClC-4 by protons and anions to quantify association of substrates with the transporter. External acidification shifts voltage dependence of ClC-4 transport to more positive potentials and leads to reduced transport currents. Internal pH changes had less pronounced effects. Uncoupled ClC-4 transport is facilitated by elevated external [SCN(-)] but impaired by internal Cl(-) and I(-). Block by internal anions indicates the existence of an internal anion-binding site with high affinity that is not present in ClC channels. The voltage dependence of ClC-4 coupled transport is modulated by external protons and internal Cl(-) in a manner similar to what is observed under uncoupling conditions. Our data illustrate functional differences but also similarities between ClC channels and transporters.
| S-EPMC3043216 | biostudies-literature
Project description:CLC proteins underlie muscle, kidney, bone, and other organ system function by catalyzing the transport of Cl(-) ions across cell and organellar membranes. Some CLC proteins are ion channels while others are pumps that exchange Cl(-) for H(+). The pathway through which Cl(-) ions cross the membrane has been characterized, but the transport of H(+) and the principle by which their movement is coupled to Cl(-) movement is not well understood. Here we show that H(+) transport depends not only on the presence of a specific glutamate residue but also the presence of Cl(-) ions. H(+) transport, however, can be isolated and analyzed in the absence of Cl(-) by mutating the glutamate to alanine and adding carboxylate-containing molecules to solution, consistent with the notion that H(+) transfer is mediated through the entry of a carboxylate group into the anion pathway. Cl(-) ions and carboxylate interact with each other strongly. These data support a mechanism in which the glutamate carboxylate functions as a surrogate Cl(-) ion, but it can accept a H(+) and transfer it between the external solution and the central Cl(-) binding site, coupled to the movement of 2 Cl(-) ions.
| S-EPMC3406864 | biostudies-literature
Project description:The ClC family of anion channels mediates the efficient, selective permeation of Cl(-) across the biological membranes of living cells under the driving force of an electrochemical gradient. In some eukaryotes, these channels are known to exhibit a unique gating mechanism, which appears to be triggered by the permeant Cl(-) anion. We infer details of this gating mechanism by studying the free energetics of Cl(-) occupancy in the pore of a prokaryotic ClC homolog. These free energetics were gleaned from 30 ns of molecular dynamics simulation on an approximately 133,000-atom system consisting of a hydrated membrane embedded StClC transporter. The binding sites for Cl(-) in the transporter were determined for the cases where the putative gating residue, Glu(148), was protonated and unprotonated. When the glutamate gate is protonated, Cl(-) favorably occupies an exterior site, S(ext), to form a queue of anions in the pore. However, when the glutamate gate is unprotonated, Cl(-) cannot occupy this site nor, consequently, pass through the pore. An additional, previously undetected, site was found in the pore near the outer membrane that exists regardless of the protonation state of Glu(148). Although this suggests that, for the prokaryotic homolog, protonation of Glu(148) may be the first step in transporting Cl(-) at the expense of H(+) transport in the opposite direction, an evolutionary argument might suggest that Cl(-) opens the ClC gate in eukaryotic channels by inducing the conserved glutamate's protonation. During an additional 20 ns free dynamics simulation, the newly discovered outermost site, S(out), and the innermost site, S(int), were seen to allow spontaneous exchange of Cl(-) ions with the bulk electrolyte while under depolarization conditions.
| S-EPMC1304573 | biostudies-literature
Project description:The first chloride transporter identified in the superfamily of ClC chloride channels was from Escherichia coli (EClC) (Accardi, A., and Miller, C. (2004) Nature 427, 803-807). Pathways, energetics, and mechanism of proton and chloride translocation and their coupling are up to now unclear. To bridge the hydrophobic gap of proton transport, we modeled four stable buried waters into both subunits of the WT EClC structure. Together they form a "water wire" connecting Glu-203 with the chloride at the central site, which in turn connects to Glu-148, the hypothetical proton exit site. Assuming the transient production of hydrochloride in the central chloride binding site of EClC, the water wire could establish a transmembrane proton transport pathway starting from Glu-203 all the way downstream onto Glu-148. We demonstrated by electrostatic and quantum chemical computations that protonation of the central chloride is energetically feasible. We characterized all chloride occupancies and protonation states possibly relevant for the proton-chloride transport cycle in EClC and constructed a working model. Accordingly, EClC evolves through states involving up to two excess protons and between one and three chlorides, which was required to fulfill the experimentally observed 2:1 stoichiometry. We show that the Y445F and E203H mutants of EClC can operate similarly, thus explaining why they exhibit almost WT activity levels. The proposed mechanism of coupled chloride-proton transport in EClC is consistent with available experimental data and allows predictions on the importance of specific amino acids, which may be probed by mutation experiments.
| S-EPMC3024792 | biostudies-literature
Project description:It has been reported that chloride-proton exchanger ClC-5 and vacuolar-type H(+)-ATPase are essential for endosomal acidification in the renal proximal cells. Here, we found that ClC-5 is expressed in the gastric parietal cells which secrete actively hydrochloric acid at the luminal region of the gland, and that it is partially localized in the intracellular tubulovesicles in which gastric H(+),K(+)-ATPase is abundantly expressed. ClC-5 was co-immunoprecipitated with H(+),K(+)-ATPase in the lysate of tubulovesicles. The ATP-dependent uptake of (36)Cl(-) into the vesicles was abolished by 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH28080), an inhibitor of H(+),K(+)-ATPase, suggesting functional expression of ClC-5. In the tetracycline-regulated expression system of ClC-5 in the HEK293 cells stably expressing gastric H(+),K(+)-ATPase, ClC-5 was co-immunoprecipitated with H(+),K(+)-ATPase, but not with endogenous Na(+),K(+)-ATPase. The SCH28080-sensitive (36)Cl(-) transporting activity was observed in the ClC-5-expressing cells, but not in the ClC-5-non-expressing cells. The mutant (E211A-ClC-5), which has no H(+) transport activity, did not show the SCH28080-sensitive (36)Cl(-) transport. On the other hand, both ClC-5 and its mutant (E211A) significantly increased the activity of H(+),K(+)-ATPase. Our results suggest that ClC-5 and H(+),K(+)-ATPase are functionally associated and that they may contribute to gastric acid secretion.
| S-EPMC3892156 | biostudies-literature
Project description:ClC-7 is a chloride channel of late endosomes and lysosomes. In osteoclasts, it may cooperate with H(+)-ATPases in acidifying the resorption lacuna. In mice and man, loss of ClC-7 or the H(+)-ATPase a3 subunit causes osteopetrosis, a disease characterized by defective bone resorption. We show that ClC-7 knockout mice additionally display neurodegeneration and severe lysosomal storage disease despite unchanged lysosomal pH in cultured neurons. Rescuing their bone phenotype by transgenic expression of ClC-7 in osteoclasts moderately increased their lifespan and revealed a further progression of the central nervous system pathology. Histological analysis demonstrated an accumulation of electron-dense material in neurons, autofluorescent structures, microglial activation and astrogliosis. Like in human neuronal ceroid lipofuscinosis, there was a strong accumulation of subunit c of the mitochondrial ATP synthase and increased amounts of lysosomal enzymes. Such alterations were minor or absent in ClC-3 knockout mice, despite a massive neurodegeneration. Osteopetrotic oc/oc mice, lacking a functional H(+)-ATPase a3 subunit, showed no comparable retinal or neuronal degeneration. There are important medical implications as defects in the H(+)-ATPase and ClC-7 can underlie human osteopetrosis.
| S-EPMC554126 | biostudies-literature