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

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Abstract
We have used the recombinant NH2-terminal myc-tagged rabbit Na+-glucose transporter (SGLT1) to study the regulation of this carrier expressed in COS-7 cells. Treatment of cells with a protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate (PMA), caused a significant decrease (38.03 ± 0.05%) in methyl α-d-glucopyranoside transport activity that could not be emulated by 4α-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 ( Vmax) was decreased from 1.29 ± 0.09 to 0.85 ± 0.04 nmol ⋅ min−1⋅ mg protein−1after PMA exposure. However, measurement of high-affinity Na+-dependent phloridzin binding revealed that there was no difference in the number of cell surface transporters after PMA treatment; maximal binding capacities were 1.54 ± 0.34 and 1.64 ± 0.21 pmol/mg protein for untreated and treated cells, respectively. The apparent sugar binding affinity (Michaelis-Menten constant) and phloridzin binding affinity (dissociation constant) were not affected by PMA. Because PKC reduced Vmaxwithout affecting the number of cell surface SGLT1 transporters, we conclude that PKC has a direct effect on the carrier, resulting in a lowering of the transporter turnover rate by a factor of two.