Abstract
A marked increase in water permeability can be induced in Xenopus oocytes by injection of mRNA from tissues that express water channels, suggesting that the water channel is a protein. In view of this and previous reports which showed that proteinases may interfere with mercurial inhibition of water transport in red blood cells (RBC), we examined the influence of trypsin, chymotrypsin, papain, pronase, subtilisin and thermolysin on water permeability as well as on ATPase activity, H +-pump, passive H + conductance, and Na +/H + exchange in apical brush-border vesicles (BBMV) and endosomal (EV) vesicles from rat renal cortex. H + transport was measured by Acridine orange fluorescence quenching and water transport by stopped-flow light scattering. AS measured by potential-driven H + accumulation in BBMV and EV, proteinase treatment had little effect on vesicle integrity. In BBMV, ecto-ATPase activity was inhibited by 15–30%, Na +/H + exchange by 20–55%, and H + conductance was unchanged. Osmotic water permeability ( P f) was 570 μ/s and was inhibited 85–90% by 0.6 mM HgCl 2; proteinase treatment did not effect P f or the HgCl 2 inhibition. In EV, NEM-sensitive H + accumulation and ATPase activity were inhibited by 95%. P f (140 μm/s) and HgCl 2 inhibition (75–85%) were not influenced by proteinase treatment. SDS-PAGE showed selective digestion of multiple polypeptides by proteinases. These results confirm the presence of water channels in BBMV and EV and demonstrate selective inhibition of ATPase function and Na +/H + exchange by proteinase digestion. The lack of effect of proteinases on water transport contrasts with previous data in RBC which showed that proteinases prevent the inhibition of water transport by mercurials. We conclude that the water channel may be a small integral membrane protein which, unlike the H +-ATPase and Na +/H + exchanger, has no functionally important membrane domains that are sensitive to proteolysis.
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