We examined the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the sodium-dependent uptake of gamma-aminobutyric acid (GABA) by the synaptosomal fraction from rat cerebral cortex. Activation of protein kinase C (PKC) by 100 nM TPA inhibited the Na(+)-dependent uptake of GABA by 38.1%, whereas 4 alpha-phorbol-12,13-didecanoate (4 alpha-PDD), an inactive phorbol ester, did not alter the uptake. The inhibition was blocked by preincubation with 100 nM staurosporine, a potent inhibitor of PKC. The Eadie-Hofstee plots revealed the presence of a high affinity uptake system. The treatment with TPA increased the Km value from 6.76 microM to 18.5 microM with a trend toward a slight decrease of Vmax. In the presence of beta-alanine, TPA inhibited the GABA uptake by increasing the Km value from 8.65 microM to 15.0 microM without affecting Vmax. The molecular basis of the inhibitory effect of TPA was further examined using Xenopus oocytes expressing GAT1, a beta-alanine-insensitive and nipecotate-sensitive neuronal GABA transporter, resulting in a similar effect of TPA. The value of Km, but not Vmax, was increased by the treatment with 100 nM TPA. These results suggest that PKC may modulate the GABA uptake into presynaptic terminals through the inhibition of GAT1 activity.