ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation.
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 alpha2-macroglobulin (alpha2M), which targets to late endosomes. In pulse label-chase experiments, [Cl-] was 19 mM just after internalization in alpha2M-labeled endosomes in primary cultures of hepatocytes from wild-type mice, increasing to 58 mM 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 alpha2M-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 alpha2M-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.
Hara-Chikuma
Mariko
M
Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521, USA.
Yang
Baoxue
B
Sonawane
N D
ND
Sasaki
Sei
S
Uchida
Shinichi
S
Verkman
A S
AS
D000143:Acids / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D018118:Chloride Channels / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D006224:Cricetinae; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism*; D017353:Gene Deletion; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D014162:Transfection
D000143:Acids; D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel