Expression of a differentiated transport function in apical membrane vesicles isolated from an established kidney epithelial cell line. Sodium electrochemical potential-mediated active sugar transport.

Electrogenic Na+ gradient-stimulated active accumulation of alpha-methyl-D-glucopyranoside was demonstrated using apical membrane vesicles isolated from an established kidney epithelial cell line (LLC-PK1). Apical membrane vesicles were purified from densely confluent cell cultures after cell disruption by nitrogen cavitation using a rapid Mg2+ differential precipitation procedure. Such preparations were 11-fold enriched in apical-specific enzyme markers, were not enriched in basolateral-specific markers, and were relatively free from contamination by intracellular membranes. Sugar accumulated within vesicles was sensitive to osmotic pressure differences across the membrane. alpha-Methyl-D-glucopyranoside transport activity in vesicles exhibited similar hexose specificities and sensitivity to inhibition by phlorizin to the corresponding transport system expressed in intact LLC-PK1 cells and in kidney proximal tubule. Vesicles prepared from subconfluent cultures, which have not yet developed the expression of this transport system, had diminished Na+-stimulated sugar transport activity yet expressed Na+-stimulated amino acid transport activity. Active accumulation as a function of increasing Na+ gradient and an interior-negative membrane potential suggested a stoichiometry between sugar and Na+ uptake of 1:2, respectively. Experimental generation of interior-negative membrane potentials in vesicles produced a further stimulation of sugar accumulation, consistent with an additive contribution from both a Na+ chemical gradient and an electrical membrane potential in energizing active sugar transport.