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(Investigative Ophthalmology and Visual Science. 2002;43:2326-2333.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Interaction of GABA Receptor/Channel {rho}1 and {gamma}2 Subunit

George M. Ekema, Wei Zheng and Luo Lu

From the Division of Molecular Medicine, Harbor-UCLA Medical Center, School of Medicine, University of California Los Angeles, Torrance, California.

PURPOSE. To determine whether protein–protein and functional interactions can occur between {gamma}-aminobutyric acid (GABA)A receptor/channels {gamma}2 subunit and the retina-specific GABAC {rho}1 subunit.

METHODS. Protein–protein interaction was characterized by immunocoprecipitation of these subunits in brain and spinal cord with anti-{gamma}2 subunit antibody and by Western blot analysis with anti-{rho}1 subunit antibody. The {rho}1 and {gamma}2 subunits were detected in the adult rat brain and spinal cord lysates that had been previously precipitated with the specific antibodies against the {rho}1 and {gamma}2 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 {gamma}2 subunit ({gamma}2-N'), the N-terminal fragment of the {rho}1 subunit ({rho}1-N'), and the full-length {rho}1 subunit, the protein–protein interaction of the GABAA {gamma}2 subunit and the GABAC {rho}1 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 {rho}1 and {gamma}2 subunits was investigated by functional studies in which {gamma}2 ({gamma}2-N' with 837 bp) and {rho}1 cRNAs were coinjected in Xenopus oocytes. In studies of the functional interaction, after injection of the {gamma}2 subunit mutant cRNA containing a N-terminal fragment, GABA-induced {rho}1 originated currents declined to 16% of the control level of homooligomeric {rho}1 current. This inhibitory effect of coexpressing {gamma}2 subunit mutants with {rho}1 subunit on the {rho}1-originated current in oocytes was dose dependent. In addition, coexpression of the GABA {rho}1 and {gamma}2 subunits in oocytes altered pharmacologic properties of the homooligomeric receptor/channel formed by {rho}1 or {gamma}2 subunits. Further evidence was provided by results obtained with specific antibodies showing that the {rho}1 subunit was coimmunoprecipitated with the {gamma}2 subunit from the retina, brain, and spinal cord.

CONCLUSIONS. The results indicate that protein–protein and functional interactions can occur between the GABAA {gamma}2 subunit and the GABAC {rho}1 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 {gamma}2 subunit and GABAC {rho}1 subunit.




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Copyright © 2002 by the Association for Research in Vision and Ophthalmology