AJP - Renal Physiology, Vol 267, Issue 6 944-F951, Copyright © 1994 by American Physiological Society

ARTICLES

Association and interactions of GTP-binding proteins with rat medullary H(+)-ATPase

N. J. Brunskill, J. J. Morrissey and S. Klahr
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri.

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.

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