Identification of Residues within GABA_AReceptor α Subunits That Mediate Specific Assembly with Receptor β Subunits

Abstract

GABA A receptors can be constructed from a range of differing subunit isoforms: ␣, ␤, ␥, ␦, and ⑀. Expression studies have revealed that production of GABA-gated channels is achieved after coexpression of ␣ and ␤ subunits. The expression of a ␥ subunit isoform is essential to confer benzodiazepine sensitivity on the expressed receptor. However, how the specificity of subunit interactions is controlled during receptor assembly remains unknown. Here we demonstrate that residues 58-67 within ␣ subunit isoforms are important in the assembly of receptors comprised of ␣␤ and ␣␤␥ subunits. Deletion of these residues from the ␣1 or ␣6 subunits results in retention of either ␣ subunit isoform in the endoplasmic reticulum on coexpression with the ␤3, or ␤3 and ␥2 subunits. Immunoprecipitation revealed that residues 58-67 mediated oligomerization of the ␣1 and ␤3 subunits, but were without affect on the production of ␣/␥ complexes. Within this domain, glutamine 67 was of central importance in mediating the production of functional ␣1␤3 receptors. Mutation of this residue resulted in a drastic decrease in the cell surface expression of ␣1␤3 receptors and the resulting expression of ␤3 homomers. Sucrose density gradient centrifugation revealed that this residue was important for the production of a 9S ␣1␤3 complex representing functional GABA A receptors.Therefore, our studies detail residues that specify GABA A receptor ␣␤ subunit interactions. This domain, which is conserved in all ␣ subunit isoforms, will therefore play a critical role in the assembly of GABA A receptors composed of ␣␤ and ␣␤␥ subunits. Key words: GABA A -receptor; assembly; cell surface expression; N-terminal; oligomerization; ␣ subunitGABA A receptors are critical mediators of fast synaptic inhibition in the brain and are also important drug targets for a range of compounds, including the benzodiazepines and barbiturates (MacDonald and Olsen, 1994;Rabow et al., 1995). GABA A receptors are members of the ligand-gated ion channel superfamily that includes glycine, nicotinic acetylcholine (AChR), and 5-HT 3 receptors (Unwin, 1993). Molecular cloning has revealed a range of GABA A receptor subunits that can be divided by homology into subunit classes with multiple members: ␣(1-6), ␤ (1-3), ␥(1-3), ␦, ⑀, and (MacDonald and Olsen, 1994;Rabow et al., 1995;Davies et al., 1997;Hedblom and Kirkness, 1997). There is considerable spatial and temporal variation in subunit expression, with many neuron types expressing multiple numbers of receptor subunits (Laurie et al., 1992;MacDonald and Olsen, 1994;Rabow et al., 1995). Clearly, to delineate the true diversity of GABA A receptor structure in the brain, it is important to gain some insights into how these receptor subunits are assembled.Studies using heterologous expression focusing on the receptor ␣1, ␤1-2, and ␥2 subunits, have revealed that access to the cell surface is limited to the combinations ␣␤ and ␣␤␥2 (Angelotti and MacDonald 1993;MacDonald and Olsen, 1994;Rabow et al., 1995;Connolly et al., 1996). Most s...

Cloning and Characterization of a GABAA Receptor γ2 Subunit Variant

“…6, A and B). These results are consistent with published results that the combination of ␣ and ␤ subunits is sufficient for creating the GABA-binding sites and that a third component, the ␥ subunit, is required for formation of the benzodiazepinebinding sites (12,25,26 (Fig. 7B) (Fig.…”

“…Failure of ␥ 2 XL to Localize to the Cell Surface-Assembly and cell-surface expression of GABA A receptor ␣␤ or ␣␤␥ heteromers have previously been described (12,25,26). Our observation that overexpression of ␥ 2 XL decreased GABA A receptor binding (Fig.…”

Alternate Use of Distinct Intersubunit Contacts Controls GABAAReceptor Assembly and Stoichiometry

“…Whereas the (ϩ) and the (✥) sites are located at the same side and are possibly situated closely together, the (Ϫ) site is located at the other side of the subunit. The recently identified sequence ␣ 1 (58 -67), which mediates binding to ␤ 3 subunits (Taylor et al, 2000), seems to form part of the (Ϫ) site because one of its residues (␣ 1 F64; Smith and Olsen, 1994) contributes to the GABA binding site assumed to be located at the ␣ 1 -␤ 3 interface of GABA A receptors (Fig. 7).…”

A Novel Site on γ3 Subunits Important for Assembly of GABAA Receptors

“…For the GABA A receptor, specific amino acid sequences have been identified in this domain that seem to mediate heteromeric and homomeric assembly of ␤ subunits (29) or assembly of ␣ with ␤ subunits (30). In addition, the ␥ 2 amino acid sequences ␥ 2 (91-104) and ␥ 2 (83-90) were identified as sites important for assembly with ␣ 1 and ␤ 3 subunits, respectively (31).…”