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 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 alpha-D-glucopyranoside transport activity that could not be emulated by 4alpha-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-1 after 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 Vmax without 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.
D000818:Animals; D019556:COS Cells / Q000378:metabolism*; D018118:Chloride Channels / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D007211:Indoles / Q000494:pharmacology; D007422:Intestines / Q000737:chemistry*; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000235:genetics*; D008757:Methylglucosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D010695:Phlorhizin / Q000378:metabolism; D015221:Potassium Channels / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D051269:Sodium-Glucose Transporter 1; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection