Solubilization and reconstitution of renal brush border Na+-H+ exchanger.
In order to permit future characterization and possible isolation of the Na+-H+ exchanger from the apical membrane of proximal tubular cells, studies were performed to solubilize and reconstitute this transporter. Rabbit brush border membranes were prepared by a magnesium aggregation method, solubilized with the detergent octyl glucoside, and reconstituted into artificial phospholipid vesicles. In the presence of a pH gradient (pHin 6.0, pHout 8.0), the uptake of 1 mM 22Na+ into the proteoliposomes was five- to sevenfold higher than into liposomes. Amiloride (2 mM) inhibited proton gradient-stimulated uptake of sodium by 50%. As compared to proton gradient conditions, the uptake of sodium was lower in the absence of a pH gradient but was significantly higher when the outside and inside pH was 6.0 than 8.0. The Ka for sodium in reconstituted proteoliposomes studied under pH gradient conditions was 4 mM. The uptake of sodium in proteoliposomes prepared from heat-denatured membrane proteins was significantly decreased. These studies demonstrate that proteoliposomes prepared from octyl glucoside-solubilized brush border membrane proteins and asolectin exhibit proton gradient-stimulated, amiloride-inhibitable, electroneutral uptake of sodium. The ability to solubilize and reconstitute the Na+-H+ exchanger from the apical membrane of the proximal tubule will be of value in isolating and characterizing this transporter.
Weinman
E J
EJ
Department of Internal Medicine, Pharmacology, and Physiology, University of Texas Medical School, Houston 77225.
Shenolikar
S
S
Cragoe
E J
EJ
Dubinsky
W P
WP
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008871:Microvilli / Q000378:metabolism*; D011510:Proteolipids; D011817:Rabbits; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers; D012995:Solubility
D002352:Carrier Proteins; D008081:Liposomes; D011510:Proteolipids; D017923:Sodium-Hydrogen Exchangers; C020485:proteoliposomes; D000584:Amiloride; D012964:Sodium