Details

Autor(en) / Beteiligte

• Richard Chapell
• Orlando F. Bueno
• Xavier Alvarez-Hernandez
• Lucy C. Robinson
• Nancy J. Leidenheimer

Titel

Activation of Protein Kinase C Induces γ-Aminobutyric Acid Type A Receptor Internalization in Xenopus Oocytes

Ist Teil von

• The Journal of biological chemistry, 1998-12, Vol.273 (49), p.32595

Ort / Verlag

American Society for Biochemistry and Molecular Biology

Erscheinungsjahr

1998

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• The inhibition of γ-aminobutyric acid (GABA)-gated chloride currents by the protein kinase C (PKC) activator 4β-phorbol 12-myristate 13-acetate (PMA) was investigated using recombinant human GABA A receptors expressed in Xenopus oocytes. PMA (5 n m ) reduced the GABA response in oocytes expressing the α1β2γ2L 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, α1β2(S410A), were comparably inhibited. Phorbol 12-monomyristate (PMM; 5 n m ), which does not activate PKC, did not alter the GABA response in either construct, while the PKC inhibitor calphostin C (0.5 μ m ) prevented the PMA effect. To further investigate PMA inhibition of the GABA response, a GABA A receptor α1 subunit/green fluorescent protein (GFP) chimera (α1GFP) was used to visualize GABA A receptor distribution. Similar to the wild type constructs, PMA robustly decreased GABA responses in oocytes expressing α1GFPβ2γ2L and α1GFPβ2(S410A) receptor constructs. Following PMA treatment, GFP fluorescence in the oocyte plasma membrane was decreased to approximately 45% of the pretreatment values indicating GABA A 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 α1 subunit, were used to demonstrate that PMA, but not PMM, decreases α1 subunit immunoreactivity in the plasma membrane of intact oocytes expressing the α1β2γ2L 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 through PKC-mediated phosphorylation of an unidentified protein(s) and that this contributes to the decrease in electrophysiological responses to GABA following PKC activation.