title: Chloride channel (Clc)-5 is necessary for exocytic trafficking of Na+/H+ exchanger 3 (NHE3).
abstract: ClC-5, a chloride/proton exchanger, is predominantly expressed and localized in subapical endosomes of the renal proximal tubule. Mutations of the CLCN5 gene cause Dent disease. The symptoms of Dent disease are replicated in Clcn5 knock-out mice. Absence of ClC-5 in mice is associated with reduced surface expression of NHE3 in proximal tubules. The molecular basis for this change is not fully understood. In this study, we investigated the mechanisms by which ClC-5 regulates trafficking of NHE3. Whether ClC-5-dependent endocytosis, exocytosis, or both contributed to the altered distribution of NHE3 was examined. First, NHE3 activity in proximal tubules of wild type (WT) and Clcn5 KO mice was determined by two-photon microscopy. Basal and dexamethasone-stimulated NHE3 activity of Clcn5 KO mice was decreased compared with that seen in WT mice, whereas the degree of inhibition of NHE3 activity by increasing cellular concentration of cAMP (forskolin) or Ca(2+) (A23187) was not different in WT and Clcn5 KO mice. Second, NHE3-dependent absorption of HCO(3)(-), measured by single tubule perfusion, was reduced in proximal tubules of Clcn5 KO mice. Third, by cell surface biotinylation, trafficking of NHE3 was examined in short hairpin RNA (shRNA) plasmid-transfected opossum kidney cells. Surface NHE3 was reduced in opossum kidney cells with reduced expression of ClC-5, whereas the total protein level of NHE3 did not change. Parathyroid hormone decreased NHE3 surface expression, but the extent of decrease and the rate of endocytosis observed in both scrambled and ClC-5 knockdown cells were not significantly different. However, the rates of basal and dexamethasone-stimulated exocytosis of NHE3 were attenuated in ClC-5 knockdown cells. These results show that ClC-5 plays an essential role in exocytosis of NHE3.
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Lin
authors.forename: Shi
authors.initials: S
authors.lastname: Jin
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Duan
authors.forename: Tong
authors.initials: T
authors.lastname: Wang
authors.forename: Sabrina
authors.initials: S
authors.lastname: Martini
authors.forename: Phuson
authors.initials: P
authors.lastname: Hulamm
authors.forename: Boyoung
authors.initials: B
authors.lastname: Cha
authors.forename: Ann
authors.initials: A
authors.lastname: Hubbard
authors.forename: Mark
authors.initials: M
authors.lastname: Donowitz
authors.forename: Sandra E
authors.initials: SE
authors.lastname: Guggino
mesh_terms: D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D002460:Cell Line; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D057973:Dent Disease / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D004705:Endocytosis / Q000187:drug effects / Q000235:genetics; D005089:Exocytosis; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009893:Opossums; D010281:Parathyroid Hormone / Q000494:pharmacology; D021381:Protein Transport / Q000235:genetics; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*
chemical_list: D001639:Bicarbonates; C471345:CLC5 protein, mouse; D018118:Chloride Channels; D010281:Parathyroid Hormone; C000613385:SLC9A3 protein, human; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers