A V-ATPase drives active, electrogenic and Na+-independent Cl- absorption across the gills of Eriocheir sinensis

Abstract

Using biochemical and electrophysiological techniques, we have examined the proposal that an H+-ATPase is involved in Cl- uptake across the gills of the Chinese crab Eriocheir sinensis. Bafilomycin A1 (1 µmol l-1), a specific inhibitor of V-ATPases, was used to investigate the importance of this H+-translocating enzyme in Cl- transport across the gill. In homogenates of ion-transporting posterior gills, we found the activity of a bafilomycin-sensitive V-ATPase to be markedly higher than in the anterior gills, which are not involved in ion transport. A similar distribution was found for the Na+/K+- and the mitochondrial F1Fo-ATPase. After differential and density centrifugation, the specific activity of the V-ATPase was enriched by a factor of 5. Neither Na+/K+- and F1Fo-ATPase activities nor acid phosphatase activity copurified with the bafilomycin-sensitive ATPase activity, indicating that at least the major portion of V-ATPase activity is not of basolateral, mitochondrial or lysosomal origin. In fluorescence studies, using Acridine Orange or Oxonol V as dyes, membrane vesicles displayed ATP-dependent proton transport and membrane potential generation, which were markedly reduced in the presence of bafilomycin. In addition to these biochemical studies, we mounted split lamellae of posterior gills in an Ussing-type chamber and measured the negative short-circuit current (Isc), which was shown to reflect active, electrogenic, Na+-independent and ouabain-insensitive Cl- absorption. After the addition of 1 µmol l-1 bafilomycin to the external bath, this Isc was reduced to about 50­60 % of its original value. Concomitantly, the conductance of the preparation decreased by about 13 %. From these results, we conclude that an apical V-ATPase drives electrogenic Cl- uptake across the posterior gills of the Chinese crab.