Functional colocalization of water channels and proton pumps in endosomes from kidney proximal tubule.

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Abstract
The apical membrane of mammalian proximal tubule undergoes rapid membrane cycling by exocytosis and endocytosis. Osmotic water and ATP-driven proton transport were measured in endocytic vesicles from rabbit and rat proximal tubule apical membrane labeled in vivo with the fluid phase marker fluorescein-dextran. Osmotic water permeability (Pf) was determined from the time course of fluorescein-dextran fluorescence after exposure of endosomes to an inward osmotic gradient in a stopped-flow apparatus. Pf was 0.009 (rabbit) and 0.029 cm/s (rat) (23.degree.C) and independent of osmotic gradient size. Pf in rabbit endosomes was inhibited reversibly by HgCl2 (KI = 0.2 mM) and had an activation energy of 6.4 .+-. 0.5 kcal/mol (15-35.degree.C). Endosomal proton ATPase activity was measured from the time course of internal pH, measured by fluorescein-dextran fluorescence, after the addition of external ATP. Endosomes contained and ATP-driven proton pump that was sensitive to N-ethylmaleimide and insensitive to vanadate and oligomycin. In response to saturating [ATP] the pump acidified the enternal pH of 7.4, the steady-state pH was 6.4 (rat) and 6.5 (rabbit). To examine whether water channels and the proton ATPase were present in the same endosome, the time course of fluorescein-dextrin fluorescence was measured in response to an osmotic gradient in the presence and absence of ATP. ATP did not alter endosome Pf, but decreased the amplitude of the fluorescence signal by 43 .+-. 3% (rabbit) and 47 .+-. 4% (rat). The signal decrease was reversed by nigericin, which collapsed the ATP-dependent pH gradient, indicating that the water channel and proton ATPase were localized to the same endosome. In addition, there was no evidence for the presence of a sodium/proton exchanger or a sodium/glucose cotransporter in these endosomes even though both proteins are known to be present in the apical membrane of proximal tubule cells. These findings demonstrate the selective retrieval of water channels and proton pumps from the apical membrane of mammalian proximal tubule epithelia into an endosomal compartment capable of maintaining an acidic pH.