ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation

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

Research output: Contribution to journalArticle

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Abstract

We investigated the involvement of ClC-3 chloride channels in endosomal acidification by measurement of endosomal pH and chloride concentration [Cl -] in control versus ClC-3-deficient hepatocytes and in control versus ClC-3-transfected Chinese hamster ovary cells. Endosomes were labeled with pH or [Cl-]-sensing fluorescent transferrin (Tf), which targets to early/recycling endosomes, or α2-macroglobulin (α2M), which targets to late endosomes. In pulse label-chase experiments, [Cl-] was 19 mM just after internalization in α2M-labeled endosomes in primary cultures of hepatocytes from wild-type mice, increasing to 58 nM over 45 min, whereas pH decreased from 7.1 to 5.4. Endosomal acidification and [Cl-] accumulation were significantly impaired in hepatocytes from ClC-3 knock-out mice, with [Cl -] increasing from 16 to 43 mM and pH decreasing from 7.1 to 6.0. Acidification and Cl- accumulation were blocked by bafilomycin. In Tf-labeled endosomes, [Cl-] was 46 mM in wild-type versus 35 mM in ClC-3-deficient hepatocytes at 15 min after internalization, with corresponding pH of 6.1 versus 6.5. Approximately 4-fold increased Cl- conductance was found in α2M-labeled endosomes isolated from hepatocytes of wild-type versus ClC-3 null mice. In contrast, Golgi acidification was not impaired in ClC-3-deficient hepatocytes. In transfected Chinese hamster ovary cells expressing ClC-3A, endosomal acidification and [Cl-] accumulation were enhanced. [Cl-] in α2M-labeled endosomes was 42 mM (control) versus 53 mM (ClC-3A) at 45 min, with corresponding pH 5.8 versus 5.2; [Cl-] in Tf-labeled endosomes at 15 min was 37 mM (control) versus 49 mM (ClC-3A) with pH 6.3 versus 5.9. Our results provide direct evidence for involvement of ClC-3 in endosomal acidification by Cl- shunting of the interior-positive membrane potential created by the vacuolar H+ pump.

Original languageEnglish
Pages (from-to)1241-1247
Number of pages7
JournalJournal of Biological Chemistry
Volume280
Issue number2
DOIs
Publication statusPublished - 2005 Jan 14
Externally publishedYes

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Acidification
Endosomes
Chlorides
Macroglobulins
Hepatocytes
Transferrin
Cricetulus
Cells
Proton Pumps
Ovary
ClC-3 channel
Recycling
Labels
Knockout Mice
Membrane Potentials
Membranes
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation. / Chikuma, Mariko; Yang, Baoxue; Sonawane, N. D.; Sasaki, Sei; Uchida, Shinichi; Verkman, A. S.

In: Journal of Biological Chemistry, Vol. 280, No. 2, 14.01.2005, p. 1241-1247.

Research output: Contribution to journalArticle

Chikuma, Mariko ; Yang, Baoxue ; Sonawane, N. D. ; Sasaki, Sei ; Uchida, Shinichi ; Verkman, A. S. / ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 2. pp. 1241-1247.
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AU - Verkman, A. S.

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