Human placental brush-border membrane Na(+)-biotin cotransport.

Membrane transport pathways for transplacental transfer of the water-soluble vitamin biotin were investigated by assessing the possible presence of a Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells. The presence of Na(+)-biotin cotransport was determined from radiolabeled tracer flux measurements of biotin uptake using preparations of purified brush-border membrane vesicles. The imposition of an inwardly directed Na+ gradient stimulated vesicle uptake of biotin to levels approximately 25-fold greater than those observed at equilibrium. The voltage sensitivity of Na+ gradient-driven biotin uptake suggested Na(+)-biotin cotransport is electrogenic occurring with net transfer of positive charge. A kinetic analysis of the activation of biotin uptake by increasing Na+ was most consistent with an interaction of Na+ at 2 sites in the transport protein. Static head determinations used to identify the magnitude of opposing driving forces bringing flux through the cotransport mechanism to equilibrium indicated a coupling ratio of 2 Na+ per biotin. Substrate specificity studies using chemical analogues of biotin suggested both the terminal carboxylic acid of the valeric acid side chain and a second nucleus of anionic charge were important determinants for substrate interaction with the cotransport protein. Initial rate determinations of biotin uptake indicate biotin interacts with a single saturable site (Km = 21 microM) with a maximal transport rate of 4.5 nmol/mg/min. The results of this study provide evidence for an electrogenic Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells.