Activation of protein kinase C induces gamma-aminobutyric acid type a receptor internalization in Xenopus oocytes

The inhibition of gamma-aminobutyric acid (GABA)-gated chloride currents by the protein kinase C (PKC) activator 4 beta-phorbol 12-myristate 13-acetat e (PMA) was investigated using recombinant human GABA(A) receptors expresse d in Xenopus oocytes, PMA (5 nM) reduced the GABA response in oocytes expre ssing the alpha 1 beta 2 gamma 2L receptor construct, as measured by the tw o-electrode voltage-clamp method. GABA responses declined to approximately 25% of their pretreatment value within 45 min. GABA responses in oocytes ex pressing a receptor con struct from which the known PKC phosphorylation sit es were absent, alpha 1 beta 2(S410A), were comparably inhibited. Phorbol 1 2-monomyristate (PMM; 5 nM), which does not activate PKC, did not alter the GABA response in either construct, while the PKC inhibitor calphostin C (0 .5 mu M) prevented the PMA effect. To further investigate PMA inhibition of the GABA response, a GABA(A) receptor alpha 1 subunit/green fluorescent pr otein (GFP) chimera (alpha 1GFP) was used to visualize GABA(A) receptor dis tribution. Similar to the wild type constructs, PMA robustly decreased GABA responses in oocytes expressing alpha 1GFP beta 2 gamma 2L and alpha 1GFP beta 2(S410A) receptor constructs. Following PMA treatment, GFP fluorescenc e in the oocyte plasma membrane was decreased to approximately 45% of the p retreatment values indicating GABA(A) receptor internalization. This effect of PMA was prevented by calphostin C and was not produced by PMM, Experime nts with bd24, a monoclonal antibody which recognizes an extracellular epit ope of the alpha 1 subunit, were used to demonstrate that PMA, but not PMM, decreases alpha 1 subunit immunoreactivity in the plasma membrane of intac t oocytes expressing the alpha 1 beta 2 gamma 2L construct, thus confirming the results obtained with the chimeric receptor. It is concluded that, in Xenopus oocytes, PMA induces an internalization of the GABA(A) receptor thr ough PKC-mediated phosphorylation of an unidentified protein(s) and that th is contributes to the decrease in electrophysiological responses to GABA fo llowing PKC activation.