PKC regulates turnover rate of rabbit intestinal Na+-glucose transporter expressed in COS-7 cells

We have used the recombinant NH2-terminal myc-tagged rabbit Na+-glucose tra nsporter (SGLT1) to study the regulation of this carrier expressed in COS-7 cells. Treatment of cells with a protein kinase C (PKC) agonist, phorbol 1 2-myristate 13-acetate (PMA), caused a significant decrease (38.03 +/- 0.05 %) in methyl alpha-D-glucopyranoside transport activity that could not be e mulated by 4 alpha-phorbol 12,13-didecanoate. The decrease in sugar uptake stimulated by PMA was reversed by the PKC inhibitor bisindolylmaleimide I. The maximal rate of Na+-glucose cotransport activity (V-max) was decreased from 1.29 +/- 0.09 to 0.85 +/- 0.04 nmol.min(-1).mg protein(-1) after PMA e xposure. However, measurement of high-affinity Na+-dependent phloridzin bin ding revealed that there was no difference in the number of cell surface tr ansporters after PMA treatment; maximal binding capacities were 1.54 +/- 0. 34 and 1.64 +/- 0.21 pmol/mg protein for untreated and treated cells, respe ctively. The apparent sugar binding affinity (Michaelis-Menten constant) an d phloridzin binding affinity (dissociation constant) were not affected by PMA. Because PKC reduced V-max without affecting the number of cell surface SGLT1 transporters, we conclude that PKC has a direct effect on the carrie r, resulting in a lowering of the transporter turnover rate by a factor of two.