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George M. Ekema, Wei Zheng, Luo Lu; Interaction of GABA Receptor/Channel ρ1 and γ2 Subunit. Invest. Ophthalmol. Vis. Sci. 2002;43(7):2326-2333.
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purpose. To determine whether protein–protein and functional interactions can occur between γ-aminobutyric acid (GABA)A receptor/channels γ2 subunit and the retina-specific GABAC ρ1 subunit.
methods. Protein–protein interaction was characterized by immunocoprecipitation of these subunits in brain and spinal cord with anti-γ2 subunit antibody and by Western blot analysis with anti-ρ1 subunit antibody. The ρ1 and γ2 subunits were detected in the adult rat brain and spinal cord lysates that had been previously precipitated with the specific antibodies against the ρ1 and γ2 subunits, respectively. A two-microelectrode voltage clamp was used to measure GABA-induced currents in oocytes. In addition, a yeast two-hybrid system was used to detect the interactions of these subunits in vivo.
results. Based on yeast transformed with the N-terminal fragment of the γ2 subunit (γ2-N′), the N-terminal fragment of the ρ1 subunit (ρ1-N′), and the full-length ρ1 subunit, the protein–protein interaction of the GABAA γ2 subunit and the GABAC ρ1 subunit was found in yeast grown in triple-dropout medium (deficient in Leu, Trp, and His) and expressing the LacZ reporter gene. Interaction of the ρ1 and γ2 subunits was investigated by functional studies in which γ2 (γ2-N′ with 837 bp) and ρ1 cRNAs were coinjected in Xenopus oocytes. In studies of the functional interaction, after injection of the γ2 subunit mutant cRNA containing a N-terminal fragment, GABA-induced ρ1 originated currents declined to 16% of the control level of homooligomeric ρ1 current. This inhibitory effect of coexpressing γ2 subunit mutants with ρ1 subunit on the ρ1-originated current in oocytes was dose dependent. In addition, coexpression of the GABA ρ1 and γ2 subunits in oocytes altered pharmacologic properties of the homooligomeric receptor/channel formed by ρ1 or γ2 subunits. Further evidence was provided by results obtained with specific antibodies showing that the ρ1 subunit was coimmunoprecipitated with the γ2 subunit from the retina, brain, and spinal cord.
conclusions. The results indicate that protein–protein and functional interactions can occur between the GABAA γ2 subunit and the GABAC ρ1 subunit. Therefore, the functional role of GABA receptor/channels in the brain, retina, and spinal cord is more diversified because of the possible assembly between the GABAA γ2 subunit and GABAC ρ1 subunit.
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