Association and interactions of GTP-binding proteins with rat medullary H(+)-ATPase.
Guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) are expressed in a heterogeneous manner in the mammalian kidney. In particular, cells of the medullary collecting tubule demonstrate a complex pattern of G protein expression both between cell types and between the polarized surfaces of individual cells. Intercalated cells expressing the H(+)-ATPase are also prevalent in this nephron segment. To examine interactions between G proteins and the H(+)-ATPase, we performed immunocytochemical studies on perfusion-fixed sections of rat kidney using polyclonal anti-G protein antibodies and E11, a mouse monoclonal antibody to the 31-kDa subunit of the vacuolar H(+)-ATPase. G alpha s subunits were consistently not associated with cells containing the H(+)-ATPase in this nephron segment, whereas G alpha i-2, G alpha i-3, and G alpha q/11 were. Some intercalated cells that stained prominently for the proton pump in the apical membrane did not, however, stain for any G protein alpha-subunit. We prepared medullary membrane vesicles highly enriched for the H(+)-ATPase to examine possible functional interactions of G proteins with the H(+)-ATPase by the acridine orange method. These vesicles were also highly enriched for G protein subunits. Proton transport was significantly increased in the presence of guanosine 5'-O-(3-thiotriphosphate), and this held true in the absence of chloride. This excludes an effect on chloride conductance indirectly stimulating the H(+)-ATPase. Guanine nucleotides did not affect the proton leak of the vesicles. Thus some G proteins are associated with the H(+)-ATPase and can regulate its function; however, the particular G proteins involved remain to be identified.
Brunskill
N J
NJ
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri.
Morrissey
J J
JJ
Klahr
S
S
D000818:Animals; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D019204:GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D007150:Immunohistochemistry; D007679:Kidney Medulla / Q000201:enzymology*; D007685:Kidney Tubules, Collecting / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis; D013873:Thionucleotides / Q000494:pharmacology; D014617:Vacuoles / Q000201:enzymology
D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D019204:GTP-Binding Proteins; D006180:Proton-Translocating ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D005033:Ethylmaleimide