title: Reconstitution of cAMP-dependent protein kinase regulated renal Na+-H+ exchanger.
abstract: Studies were performed to determine if the Na+-H+ exchanger, solubilized from renal brush border membranes from the rabbit and assayed in reconstituted artificial proteoliposomes, could be regulated by cAMP-dependent protein kinase. Octyl glucoside solubilized renal apical membrane proteins from the rabbit kidney were phosphorylated by incubation with ATP and highly purified catalytic subunit of cAMP-dependent kinase. 22Na+ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. cAMP-dependent protein kinase resulted in sustained protein phosphorylation and a concentration-dependent decrease in the amiloride-sensitive component of pH gradient-stimulated sodium uptake. The inhibitory effect of cAMP-dependent protein kinase demonstrated an absolute requirement for ATP and was blocked by the specific protein inhibitor of this kinase. cAMP-dependent protein kinase also inhibited 22Na+ uptake in the absence of a pH gradient (pHin 6.0, pHout 6.0) and the inhibitory effect was blocked by the specific inhibitor of the kinase. Solubilized membrane proteins exhibited little endogenous protein kinase or protein phosphatase activity. These studies indicate that Na+-H+ exchange activity of proteoliposomes reconstituted with proteins from renal brush border membranes is inhibited by phosphorylation of selected proteins by cAMP-dependent protein kinase. These findings also indicate that the regulatory components of the Na+-H+ exchanger remain active during the process of solubilization and reconstitution of renal apical membrane proteins.
authors.affiliation: Department of Internal Medicine, Pharmacology, and Physiology, University of Texas School of Medicine, Houston 77225.
authors.forename: E J
authors.initials: EJ
authors.lastname: Weinman
authors.forename: W P
authors.initials: WP
authors.lastname: Dubinsky
authors.forename: S
authors.initials: S
authors.lastname: Shenolikar
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D006706:Homeostasis; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008871:Microvilli / Q000201:enzymology*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; D011494:Protein Kinases