PHORBOL ESTER-INDUCED INHIBITION OF GABA UPTAKE BY SYNAPTOSOMES AND BY XENOPUS-OOCYTES EXPRESSING GABA TRANSPORTER (GAT1)

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 K-m value from 6.76 mu M to 18.5 mu M with a trend toward a slight decrease of V-max. In the presence of beta-alanine, TPA inhibited the GABA uptake by increasing the K-m value from 8.65 mu M to 15.0 mu M without affecting V-max. 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 K-m, but not V-max, 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.