title: Functional vacuolar ATPase (V-ATPase) proton pumps traffic to the enterocyte brush border membrane and require CFTR.
abstract: Vacuolar ATPases (V-ATPases) are highly conserved proton pumps that regulate organelle pH. Epithelial luminal pH is also regulated by cAMP-dependent traffic of specific subunits of the V-ATPase complex from endosomes into the apical membrane. In the intestine, cAMP-dependent traffic of cystic fibrosis transmembrane conductance regulator (CFTR) channels and the sodium hydrogen exchanger (NHE3) in the brush border regulate luminal pH. V-ATPase was found to colocalize with CFTR in intestinal CFTR high expresser (CHE) cells recently. Moreover, apical traffic of V-ATPase and CFTR in rat Brunner's glands was shown to be dependent on cAMP/PKA. These observations support a functional relationship between V-ATPase and CFTR in the intestine. The current study examined V-ATPase and CFTR distribution in intestines from wild-type, CFTR(-/-) mice and polarized intestinal CaCo-2BBe cells following cAMP stimulation and inhibition of CFTR/V-ATPase function. Coimmunoprecipitation studies examined V-ATPase interaction with CFTR. The pH-sensitive dye BCECF determined proton efflux and its dependence on V-ATPase/CFTR in intestinal cells. cAMP increased V-ATPase/CFTR colocalization in the apical domain of intestinal cells and redistributed the V-ATPase Voa1 and Voa2 trafficking subunits from the basolateral membrane to the brush border membrane. Voa1 and Voa2 subunits were localized to endosomes beneath the terminal web in untreated CFTR(-/-) intestine but redistributed to the subapical cytoplasm following cAMP treatment. Inhibition of CFTR or V-ATPase significantly decreased pHi in cells, confirming their functional interdependence. These data establish that V-ATPase traffics into the brush border membrane to regulate proton efflux and this activity is dependent on CFTR in the intestine.
authors.affiliation: Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut;
authors.forename: Anne M
authors.initials: AM
authors.lastname: Collaco
authors.forename: Peter
authors.initials: P
authors.lastname: Geibel
authors.forename: Beth S
authors.initials: BS
authors.lastname: Lee
authors.forename: John P
authors.initials: JP
authors.lastname: Geibel
authors.forename: Nadia A
authors.initials: NA
authors.lastname: Ameen
mesh_terms: D000818:Animals; D018938:Caco-2 Cells; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D020895:Enterocytes / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008871:Microvilli / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D025262:Vacuolar Proton-Translocating ATPases / Q000502:physiology*
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D025262:Vacuolar Proton-Translocating ATPases
keywords: CFTR; V-ATPase; cAMP-regulated traffic; intestine