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. Gα_s subunits were consistently not associated with cells containing the H⁺-ATPase in this nephron segment, whereas Gα_i-2, Gα_1-3, and Gα_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 α-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.