Related Experiment Videos
Localization of the Na+-sugar cotransport system in a kidney epithelial cell line (LLC PK1)
Abstract:
Studies of the localization of the Na+-dependent sugar transport in monolayers of LLC PK1 cells show that the uptake of a methyl alpha-D-glucoside, a nonmetabolizable sugar which shares the glucose-galactose transport system, occurs mainly from the apical side of the monolayer. Kinetics of [3H]phlorizin binding to monolayers of LLC PK1 cells were also measured. These studies demonstrate the presence of two distinct classes of receptor sites. The class comprising high affinity binding sites had a dissociation constant (Kd) of 1.2 microM and a concentration of high affinity receptors of 0.30 mumol binding sites per g DNA. The other class involving low affinity sites had a Kd of 240 microM with the number of binding sites equal to 12 mumol/g DNA. Phlorizin binding at high affinity binding sites is a Na+-dependent process. Binding at the low affinity sites on the contrary is Na+-independent. The mode of action of Na+ on the high affinity binding sites was to increase the dissociation constant without modifying the number of binding sites. The Na+ dependence and the matching of Kd for high affinity binding sites with the Ki of phlorizin for the inhibition of methyl alpha-D-glucoside strongly suggest that the high affinity phlorizin binding site is, or is part of the methyl alpha-D-glucoside transport system. Binding studies from either side of the monolayer also show that the binding of phlorizin at the Na+ dependent high affinity binding sites occurs mainly from the apical rather than the basolateral side. The specific location of the Na+-dependent sugar transport system in the apical membrane of LLC PK1 cells is, therefore, another expression of the functional polarization of epithelial cells that is retained under tissue culture condition. In addition, since this sugar transport almost disappears after the cells are brought into suspension, it can be used as a marker to study the development of the apical membrane in this cell line.