Cytochalasin B binding to rabbit proximal tubular basolateral membranes

Abstract

Cytochalasin B binding to rabbit proximal tubular basolateral membranes. Cytochalasin B binds to the Na+-independent D-glucose transporter in non-renal tissues. We have shown previously that the Na+-independent D-glucose transporter of the rabbit renal proximal tubular cell is localized exclusively in the basolateral membrane. To determine whether cytochalasin B binds to this renal transporter we measured binding of [3H]cytochalasin B to proximal tubular basolateral membranes isolated from rabbit kidneys. A steady state of binding is reached by 15 minutes at 20°C over a concentration range of 0.01 to 50 µM. Non-linear regression analysis of cytochalasin B binding from 0.01 to 20 µM plotted according to Scatchard reveals two classes of binding sites with Kd 5.88 x 10−8M, Bmax 16.1 pmol/mg protein; and Kd 5.62 x 10−5M, Bmax 2816 pmol/mg protein. [3H]cytochalasin B (0.1 µM) binding to basolateral membranes is a reversible process; it is displacable by excess unlabeled cytochalasin B with a time course similar to binding of [3H]cytochalasin B. Binding of [3H]cytochalasin B is inhibited by 500mM D-glucose (21%), 2-deoxy-D-glucose (57%) and 3-O-methyl-D-glucose (64%), but not by L-glucose. [3H]cytochalasin B binding is reduced 71% by 0.1mM phloretin, but only 26% by 0.1mM phlorizin. Such substrate specificity and inhibitor sensitivity are similar to those previously demonstrated in non-renal tissues by others as well as in rabbit renal proximal tubular basolateral membranes by us. Our data suggest that cytochalasin B binds to the Na+-independent D-glucose transporter or a component of the transporter in the renal proximal tubular basolateral membrane