ClC-3 Chloride Channels Facilitate Endosomal Acidification and Chloride Accumulation

Hara‐Chikuma, Mariko; Yang, Baoxue; Sonawane, N.D.; Sasaki, Sei; Uchida, Shinichi; Verkman, A. S.

doi:10.1074/jbc.m407030200

Cited by 158 publications

(156 citation statements)

References 38 publications

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“…This might be related to their additional expression in other compartments [ClC-3 is also found on synaptic vesicles (5)] or to so-far-unknown functional differences. Vesicular CLC proteins may facilitate luminal acidification by shunting proton pump currents (1), as experimentally confirmed for ClC-5 (8) and -3 (5,6). Whereas the acidification of the osteoclast resorption lacuna was impaired in the ClC-7 KO (12), the lysosomal pH of Clcn7 Ϫ/Ϫ neurons was normal despite severe lysosomal storage disease (13).…”

Section: Discussionmentioning

confidence: 91%

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Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6

Poët

Kornak

Schweizer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

169

221

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Mammalian CLC proteins function as Cl ؊ channels or as electrogenic Cl ؊ ͞H ؉ exchangers and are present in the plasma membrane and intracellular vesicles. We now show that the ClC-6 protein is almost exclusively expressed in neurons of the central and peripheral nervous systems, with a particularly high expression in dorsal root ganglia. ClC-6 colocalized with markers for late endosomes in neuronal cell bodies. The disruption of ClC-6 in mice reduced their pain sensitivity and caused moderate behavioral abnormalities. Neuronal tissues showed autofluorescence at initial axon segments. At these sites, electron microscopy revealed electron-dense storage material that caused a pathological enlargement of proximal axons. These deposits were positive for several lysosomal proteins and other marker proteins typical for neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. However, the lysosomal pH of Clcn6 ؊/؊ neurons appeared normal. CLCN6 is a candidate gene for mild forms of human NCL. Analysis of 75 NCL patients identified ClC-6 amino acid exchanges in two patients but failed to prove a causative role of CLCN6 in that disease.acidification ͉ anion transport ͉ Batten disease ͉ channelopathy ͉ Kufs' disease

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Section: Discussionmentioning

confidence: 91%

“…4B). Because other vesicular CLC proteins facilitate vesicular acidification (5,6,8), we determined the lysosomal pH of cultured hippocampal neurons by ratiometric fluorescence imaging (see Fig. 10, which is published as supporting information on the PNAS web site).…”

Section: Redistribution Of Late-endosomal͞lysosomal Markers and Lysosmentioning

confidence: 99%

Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6

Poët

Kornak

Schweizer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

169

221

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“…This idea was supported by the finding of impaired endosomal acidification in ClC-3-deficient mice (20). However, the realization that ClC-3 through 7 are all Cl Ϫ /H ϩ antiporters rather than channels has raised questions regarding the role of these proteins in vesicular electrophysiology (25,52,53).…”

Section: Discussionmentioning

confidence: 99%

“…ClC-3 is required for proper acidification of synaptic vesicles (12), insulin granules (19), lysosomes (11), and endosomes (20) by the V-ATPase. This acidification process leads to intra-organellar pH values ranging from 5.9 to 6.2 for early endosomes to 5.0 -6.0 for late endosomes, and 4.6 -5.5 for lysosomes (21,22).…”

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confidence: 99%

The ClC-3 Cl−/H+ Antiporter Becomes Uncoupled at Low Extracellular pH

Matsuda

Filali

Collins

et al. 2010

Journal of Biological Chemistry

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ClC-3, -4, and -5 are Cl Ϫ /H ϩ antiporters that are expressed primarily in intracellular organelles. They have been proposed to provide shunt conductances for proton current generated by the vacuolar (V-type) H ϩ -ATPase (V-ATPase) 2 (1). Sufficient ClC-4 and -5 protein localizes to the plasma membrane to readily allow recording of ion currents when these proteins are heterologously expressed in Xenopus oocytes (2). Unfortunately, recombinant ClC-3 currents have been much more difficult to express (2-4) and interpretation of the currents observed (5-8) has been controversial (for review see Ref. 9). ClC-3 localizes to endosomes (10) and lysosomes (11) as well as secretory vesicles of various types (12)(13)(14) suggesting that the protein cycles through the plasma membrane. Membrane localization of ClC-3 was quantified in cultured fibroblasts using recombinant protein with both extracellular and intracellular epitope tags. It had a half-life in the membrane of ϳ9 min and about 6% of total ClC-3 protein localized to the membrane at a given time (15). A proportion of ClC-3-eGFP fusion protein clearly appears to localize to the plasma membrane of ClC-3 expressing HEK293 cells (8).We recently demonstrated that adenoviral-mediated overexpression of ClC-3 produced novel currents in HEK293 cells at neutral pH (8). These currents exhibited very steep outward rectification and time-dependent activation that was reminiscent of ClC-4 and ClC-5 (2, 16) except that the kinetics of activation were slower. Activation was also significantly slower than observed when much smaller currents were elicited by overexpression of ClC-3 plasmids in Chinese hamster ovary-K1 cells (11), although rectification properties and the effect of a specific mutation (E224A) were very similar. Changes in current amplitude and reversal potential observed in response to alterations in extracellular Cl Ϫ and H ϩ concentration were consistent with ClC-3 acting as a Cl Ϫ /H ϩ antiporter and deviated significantly from the behavior of an anion channel. These results supported predictions, based upon sequence homology, that like ClC-4 and ClC-5, ClC-3 would be a ClClC-3 is required for proper acidification of synaptic vesicles (12), insulin granules (19), lysosomes (11), and endosomes (20) by the V-ATPase. This acidification process leads to intra-organellar pH values ranging from 5.9 to 6.2 for early endosomes to 5.0 -6.0 for late endosomes, and 4.6 -5.5 for lysosomes (21,22). A Cl Ϫ channel is well suited to provide charge neutralization for a proton pump. However, the realization that the intracellular ClCs are Cl Ϫ /H ϩ antiporters (8,17,18) yielded the surprising requirement that protons move out of the endosome in exchange for Cl Ϫ to provide countercurrent for the V-ATPase. The 2Cl Ϫ to 1H ϩ stoichiometry of this process (23, 24) makes this mechanism feasible, but has led to speculation that the primary goal of this coupled transport system may be to concentrate Cl Ϫ in the compartment rather than to facilitate acidification (18,22). This adds an u...

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“…It is an interesting speculation whether CD95L could also target intracellular chloride channels of the ClC-type chloride channel family (ClC3-7), which have been reported to be expressed in intracellular vesicles, including endosomes (for review, see Refs. 43 and 51) and play a role in hepatocellular endosomal acidification (52).…”

Section: Discussionmentioning

confidence: 99%

Amplification of CD95 Activation by Caspase 8-induced Endosomal Acidification in Rat Hepatocytes

Reinehr

Sommerfeld

Keitel

et al. 2008

Journal of Biological Chemistry

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Although in rat hepatocytes CD95 is predominantly located inside the cell with almost undetectable immunostaining at the plasma membrane, the addition of CD95-ligand (CD95L) induces hepatocyte apoptosis, which is preceded by a targeting and activation of intracellularly localized CD95 to the plasma membrane including formation of the death-inducing signaling complex. This process involves an NADPH oxidase-dependent generation of reactive oxygen species (ROS) through a ceramide-and protein kinase C-dependent pathway, which leads to an activating phosphorylation of p47 phox . The mechanisms underlying CD95L-induced ceramide formation were addressed in the present study. It was found that CD95L lowered within seconds the apparent vesicular pH from 6.0 to 5.7 in a fluorescein isothiocyanate-dextran-accessible endosomal compartment, which was previously shown to contain acidic sphingomyelinase, and decreased N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide fluorescence, suggestive for an increase of cytosolic [Cl ؊ ]. Bafilomycin or 4,4-diisothiocyanostilbene-2,2-disulfonic acid disodium salt largely abolished the CD95L-induced endosomal acidification, ceramide formation, and downstream events, such as p47 phox phosphorylation, ROS formation, CD95 activation, and apoptosis. These responses were also abolished after knock-down of acidic sphingomyelinase in rat hepatocytes. Interestingly, caspase 8 inhibitors abolished these CD95L-induced signaling events, including the increase in cytosolic [Cl ؊ ], endosomal acidification, ceramide formation, and ROS generation as well as CD95 targeting to the plasma membrane and CD95 activation. The data suggest that CD95L initiates a rapid caspase 8-dependent endosomal acidification, which triggers ceramide-dependent ROS formation as an upstream event of trafficking of intracellularly stored CD95 to the plasma membrane. It is concluded that a rapid caspase 8 activation in response to CD95L signals to intracellularly stored CD95, which becomes activated and targeted to the plasma membrane. This autoamplification of CD95-activation is required for apoptosis induction. CD952 (Apo-1/Fas) belongs to the death receptor family and plays an important role in apoptosis induction in many cell types. In hepatocytes, CD95 is mainly located within the cellular interior and can either be activated after ligation with its natural ligand (CD95L) or in a ligand-independent way by hydrophobic bile salts or hyperosmotic cell shrinkage (Refs. 1-4; for review, see Ref. 5). Ligand-dependent and -independent CD95 activation in hepatocytes is a complex process that finally results in CD95 trafficking from the cellular interior to the plasma membrane, subsequent formation of the death-inducing signaling complex (DISC), and eventually apoptosis (1-12).In hepatocytes, proapoptotic stimuli such as CD95 ligand (CD95L), hydrophobic bile salts, or hyperosmotic cell shrinkage induce a rapid oxidative stress (ROS) response (2, 4), which triggers a Yes-dependent but ligand-independent activation of the epiderm...

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ClC-3 Chloride Channels Facilitate Endosomal Acidification and Chloride Accumulation

Abstract: We investigated the involvement of ClC-3 chloride channels in endosomal acidification by measurement of endosomal pH and chloride concentration [

Cited by 158 publications

References 38 publications

Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6

Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6

The ClC-3 Cl−/H+ Antiporter Becomes Uncoupled at Low Extracellular pH

Amplification of CD95 Activation by Caspase 8-induced Endosomal Acidification in Rat Hepatocytes

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