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 γ-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 4α-phorbol-12,13-didecanoate (4α-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 μM to 18.5 μM with a trend toward a slight decrease of V_max. In the presence of β-alanine, TPA inhibited the GABA uptake by increasing the K_m value from 8.65 μM to 15.0 μM without affecting V_max. The molecular basis of the inhibitory effect of TPA was further examined using Xenopus oocytes expressing GAT1, a β-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.