Regulation of the human vitamin C transporters expressed in COS-1 cells by protein kinase C [corrected

Abstract

Protein kinase C (PKC) regulation of l-ascorbic acid transport mediated by the Na+/ascorbic acid transporters, hSVCT1 and hSVCT2, expressed in COS-1 cells was studied using recombinant carboxyl-terminal V5 epitope-tagged forms of the transporters. The PKC activator phorbol 12-myristate 13-acetate (PMA) caused a time-dependent and concentration-dependent decrease (40-60%) in ascorbic acid transport activity. Effects of PMA were not observed with the inactive phorbol ester 4 alpha-phorbol and were reversed by treatment of the cells with the PKC-specific inhibitor Ro-31-8220. Kinetically, the reduction in hSVCT1 and hSVCT2 activity arose from a decrease in maximal velocity with no change in the apparent affinity. Western blot and confocal microscopy analyses indicated that the total pool of hSVCT1 or hSVCT2 proteins expressed in the transfected COS-1 cells remained unaffected by PMA treatment. For hSVCT1 the decrease in L-ascorbic acid correlated with a redistribution of the transporter from the cell surface to intracellular membranes. However, for hSVCT2 there was no apparent change in transporter distribution, suggesting that the PKC-dependent modulation of L-ascorbic acid transport mediated by hSVCT2 was the result of reduced catalytic transport efficiency.