J Biol Chem 1998 Dec 04;27349:32595-601.

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

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The inhibition of gamma-aminobutyric acid (GABA)-gated chloride currents by the protein kinase C (PKC) activator 4beta-phorbol 12-myristate 13-acetate (PMA) was investigated using recombinant human GABAA receptors expressed in Xenopus oocytes. PMA (5 nM) reduced the GABA response in oocytes expressing the alpha1 beta2 gamma2L receptor construct, as measured by the two-electrode voltage-clamp method. GABA responses declined to approximately 25% of their pretreatment value within 45 min. GABA responses in oocytes expressing a receptor construct from which the known PKC phosphorylation sites were absent, alpha1 beta2(S410A), were comparably inhibited. Phorbol 12-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 microM) prevented the PMA effect. To further investigate PMA inhibition of the GABA response, a GABAA receptor alpha1 subunit/green fluorescent protein (GFP) chimera (alpha1GFP) was used to visualize GABAA receptor distribution. Similar to the wild type constructs, PMA robustly decreased GABA responses in oocytes expressing alpha1GFP beta2 gamma2L and alpha1GFP beta2(S410A) receptor constructs. Following PMA treatment, GFP fluorescence in the oocyte plasma membrane was decreased to approximately 45% of the pretreatment values indicating GABAA receptor internalization. This effect of PMA was prevented by calphostin C and was not produced by PMM. Experiments with bd24, a monoclonal antibody which recognizes an extracellular epitope of the alpha1 subunit, were used to demonstrate that PMA, but not PMM, decreases alpha1 subunit immunoreactivity in the plasma membrane of intact oocytes expressing the alpha1 beta2 gamma2L construct, thus confirming the results obtained with the chimeric receptor. It is concluded that, in Xenopus oocytes, PMA induces an internalization of the GABAA receptor through PKC-mediated phosphorylation of an unidentified protein(s) and that this contributes to the decrease in electrophysiological responses to GABA following PKC activation.

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Species referenced: Xenopus laevis
Genes referenced: gabarap