Abstract
SummaryIt 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 36Cl− 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 36Cl− 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 36Cl− 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.
Highlights
ClC-5 belongs to a sub-branch of CLC family of Cl2 channels and transporters that includes ClC-3 and ClC-4 (Jentsch et al, 2005; Jentsch, 2008)
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
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
Summary
ClC-5 belongs to a sub-branch of CLC family of Cl2 channels and transporters that includes ClC-3 and ClC-4 (Jentsch et al, 2005; Jentsch, 2008). ClC-5 is predominantly expressed in the kidney, and its expression largely overlaps with the vacuolar-type H+-ATPase (V-ATPase), which is highly expressed in renal proximal tubules (Gunther et al, 1998). The endosomal ClC-5 is essential for proximal tubular endocytosis as evidenced by results with the ClC-5 knock-out mice, a model for Dent’s disease (Piwon et al, 2000; Wang et al, 2000). ClC-5 was previously suggested to be an electrically shunting Cl2 channel in early endosomes, facilitating intraluminal acidification (Gunther et al, 2003; Piwon et al, 2000)
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