Purpose. The inhibitory neuroiransmitter y-aminobutyric acid (GABA) activates two types of chloride channel receptors, named GABAA and GABA,-. GABAA receptors are pentamers composed of a, β, y and 0 subunits, whereas GABAf- receptors appear to be formed of p subunits. However, rod bipolar cells in rat retina exhibiting GABA,-. responses contain a, βand p subunits, raising the possibility that G ABAç receptors may be actually formed of a mixture of subunits of each type. In this study, functional and protein-based approaches were used to examine whether p and GABAA subunits coassemble. Methods. Four mutants were constructed: N-p 1 contains the N-terminal half of pi up to codon 256, and C-pl contains the signal peptide and the C-terminal half; the plβl chimera contains the N-terminus of p 1 and the C-terminus of β1, and the βl p l chimera contains the N-terminus of β1 and the C-terminus of p 1. Wild-type and mutant GABA p and GABAA subunits were translated in vitro using rabbit reticulocyte lysate and canine pancreatic microsomes (Promega). For functional analysis, cRNA was synthesized, injected into Xenopus oocytes and current amplitudes were recorded. Results. Sequential immunoprecipitation of in vitro translated subunits with subunit-specific antibodies demonstrated that pi and p2 interacted, implying that they form heterooligomeric GABA receptors in vivo. The specific co-immunoprecipitation of N-p 1 with p2 in viiro indicated that the N-terminus contains the signals for heterooligomeric p receptor assembly. However, neither pi, N-pl or p2 interacted with the GABAA subunits ccl, a5 or βl. We speculated that the lack of interaction was the result of different recognition signals in the N-termini of p and GABAA subunits. To test this hypothesis, additional experiments were performed using chimeric subunits. The βlpl chimeric subunit co-immunoprecipitated with al, but did not interact with fulllength pi. Conversely, the plβl chimera did not immunoprecipitate with ctl.but instead interacted with p 1. hi addition, plβl interfered with p l receptor formation in Xenopus oocytes, implying its interaction and assembly with p 1. Conclusion. The N-termini of p subunits and G AB AA subunits contain different recognition signals that facilitate their selective assembly into GABA,. or GABA. receptors, respectively.
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Dec 1 1997|
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