Serine 171, a Conserved Residue in the (cid:1) -Aminobutyric Acid Type A (GABA A ) Receptor (cid:1) 2 Subunit, Mediates Subunit Interaction and Cell Surface Localization*

Serine 171 in the GABA A receptor (cid:1) 2 subunit is highly conserved in the ligand-gated ion channel superfamily. In this paper, we report that mutating serine 171 within (cid:1) 2 to glycine or cysteine prevents the interaction of (cid:1) 2 with (cid:2) 2 and (cid:3) 1 when these subunits are co-expressed in human embryo kidney 293 cells, resulting in intracellular retention of (cid:1) 2. Structure analysis based on a three-dimensional homology model of (cid:1) 2 (Ernst, M., Brauchart, D., Boresch, S., and Sieghart, W. (2003) Neuroscience 119, 933–943) reveals that serine 171 may play a critical role in the formation and stabilization of an exposed turn structure that is part of the subunit interaction site. Mutation of serine 171 in the (cid:1) 2 subunit could therefore result in alteration of the structure of the subunit interaction site, preventing correct subunit assembly. (cid:1) site-directed mu- tagenesis kit (Stratagene) was used for creating the single amino acid substitution. The following amino acid substitutions were made in the mature (cid:1) 2 subunit: E168I, F169I, S170G, S171C, S171G, Y172C, Y172F, G173A, and P175T. Identical sets of mutations were made in the (cid:1) 2 and (cid:1) 2 Myc constructs, respectively. The (cid:1) 2 mutants generated were verified by double strand DNA sequencing. Cell Cultures and Transfection— Human embryonic kidney 293 cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Invitrogen) and transfected using LipofectAMINE 2000 (Invitrogen). Transfection was performed with equal molar concentra- tions of the subunit constructs either in 10-cm dishes with 24 (cid:4) g of plasmid DNA per dish or in 6-well plates with 5 (cid:4) g/well plasmid DNA. Radioligand Binding— Cells were harvested 48 h after co-transfec-tion with the (cid:2) 2, (cid:3) 1, and either the wild-type (cid:1) 2 or one of the (cid:1) 2 mutant constructs. To prepare the membrane fraction, cells were homogenized in ice-cold wash buffer (10 m M potassium phosphate, pH 7. 2) by passing through a 27-gauge needle. Cell homogenates were washed by two

Serine 171 in the GABA A receptor ␥2 subunit is highly conserved in the ligand-gated ion channel superfamily. In this paper, we report that mutating serine 171 within ␥2 to glycine or cysteine prevents the interaction of ␥2 with ␣2 and ␤1 when these subunits are co-expressed in human embryo kidney 293 cells, resulting in intracellular retention of ␥2. Structure analysis based on a three-dimensional homology model of ␥2 (Ernst, M., Brauchart, D., Boresch, S., and Sieghart, W. (2003) Neuroscience 119, 933-943) reveals that serine 171 may play a critical role in the formation and stabilization of an exposed turn structure that is part of the subunit interaction site. Mutation of serine 171 in the ␥2 subunit could therefore result in alteration of the structure of the subunit interaction site, preventing correct subunit assembly.
␥-Aminobutyric acid type A (GABA A ) 1 receptors are members of the ligand-gated ion channel superfamily, which also includes nicotinic acetylcholine (ACh), glycine, glutamate, and 5-hydroxytryptamine type 3 (5HT3) receptors. Members of this superfamily share significant sequence similarity, implying a common evolutionary origin (2).
GABA A receptors are the major inhibitory receptors in the brain and are the targets of clinically important drugs such as benzodiazepines, barbiturates, neurosteroids, and anesthetics (3). Several distinct classes of GABA A receptor subunits have been identified (3)(4)(5)(6)(7)(8). Heterologous expression of recombinant GABA A receptor subunits in cultured cells demonstrates that the combination of ␣ and ␤ subunits is sufficient for creating the GABA binding sites to elicit GABA-gated Cl Ϫ currents (9). Benzodiazepines bind to a distinct site on the GABA A receptor and positively modulate GABA-gated C Ϫ current (10). Formation of the benzodiazepine binding site requires the presence of a third component, the ␥ subunit (9,10). Typical native GABA A receptors have been proposed to be pentameric transmembrane proteins formed by two ␣, two ␤, and one ␥ subunits (9). Assembly of the hetero-pentamers occurs in the endoplasmic reticulum and requires the interaction between subunit-specific contact sites. Access to the cell surface is restricted to the correctly assembled GABA A receptors (11)(12)(13)(14)(15).
We identified previously two highly conserved residues, serine 171 and tyrosine 172, in the ␥2 subunit. These two residues flank a novel alternative exon (16,17). When the residues are mutated, oligomerization of ␥2 with ␣2 and ␤1 is prevented, and cell surface access of ␥2 is abolished. In the present study, using single amino acid substitutions, we further demonstrate that serine 171 within ␥2 plays a critical role in mediating the subunit interaction and cell surface expression of ␥2, whereas tyrosine 172, as well as other residues surrounding Ser-171/ Tyr-172, exhibit little or no effect on the function of the ␥2 subunit. Mapping of the ␥2 protein sequence onto the threedimensional homology model of the extracellular component of the GABA A receptor generated from the ACh-binding protein (AChBP) structure (1,18) suggests that Ser-171 links a ␤-strand (␤7) to a loop structure (loop 8) of ␥2. Serine 171 presumably stabilizes the loop turn by forming a hydrogen bond via its side-chain hydroxyl group. Furthermore, sidechain packing constraints may sterically exclude other amino acids at this position. This loop turn is located on the surface of a ␤-rich structure in the extracellular domain of ␥2 and is likely involved in oligomerization with other receptor subunits.

EXPERIMENTAL PROCEDURES
Plasmid Constructs-Preparation of the cDNA constructs expressing the human ␣2, ␤1, and ␥2 subunits, with or without epitope tags, have been described previously (16). Briefly, the ␣2 and ␤2 cDNAs were subcloned into the pCEP4 expression vector (Invitrogen) and used for ligand binding and immunostaining assays. To make v5-tagged constructs for co-precipitation assays, the ␣2 and ␤1 cDNAs were subcloned into the pcDNA3.1 vector (Invitrogen) with a v5 tag inserted before the stop codon. The ␥2 cDNA was subcloned into the pcDNA3.1 vector and used for ligand binding assays. The Myc-tagged ␥2 (␥2 myc ), which contains a myc-His tag at the 3Ј-terminus and a second myc tag inserted between the ninth and tenth residues of the mature protein, was also subcloned into the pcDNA3.1 vector and used for immunostaining and co-precipitation assays.
Mutagenesis-The ␥2 and ␥2 Myc cDNAs in the pcDNA3.1 vector were used for mutagenesis experiments. QuikChange II site-directed mutagenesis kit (Stratagene) was used for creating the single amino acid substitution. The following amino acid substitutions were made in the mature ␥2 subunit: E168I, F169I, S170G, S171C, S171G, Y172C, Y172F, G173A, and P175T. Identical sets of mutations were made in the ␥2 and ␥2 Myc constructs, respectively. The ␥2 mutants generated were verified by double strand DNA sequencing.
Cell Cultures and Transfection-Human embryonic kidney 293 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Invitrogen) and transfected using LipofectAMINE 2000 (Invitrogen). Transfection was performed with equal molar concentrations of the subunit constructs either in 10-cm dishes with 24 g of plasmid DNA per dish or in 6-well plates with 5 g/well plasmid DNA.
Radioligand Binding-Cells were harvested 48 h after co-transfection with the ␣2, ␤1, and either the wild-type ␥2 or one of the ␥2 mutant constructs. To prepare the membrane fraction, cells were homogenized in ice-cold wash buffer (10 mM potassium phosphate, pH 7. 2) by passing through a 27-gauge needle. Cell homogenates were washed by two * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: Incyte Corporation, 3160 Porter Dr., Palo Alto, CA 94304. Tel.: 650-621-8639; E-mail: pjin@incyte.com. 1 The abbreviations used are: GABA A , ␥-aminobutyric acid type A; 293 cells, human embryonic kidney 293 cells; ACh, acetylcholine; AChBP, ACh-binding protein; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline. centrifugation-resuspension cycles in 1 ml of ice-cold wash buffer. Cell homogenates were centrifuged at 4°C for 60 min at 15,000 ϫ g. The pellets were resuspended in 600 l of ice-cold assay buffer (10 mM potassium phosphate, pH 7, and 0.02 mM potassium chloride) and centrifuged at 500 ϫ g for 5 min. The supernatant, which contained the enriched membrane fraction, was collected and stored at Ϫ80°C after its protein concentration was determined. For radioligand binding, 150 -200 g of membrane protein preparation was incubated on ice for 60 min with 10 nM of either [ 3 H]Ro15-1788 (78 Ci/mmol; PerkinElmer Life Sciences) or [ 3 H]muscimol (28. 5 Ci/mmol; PerkinElmer Life Sciences) in a total volume of 200 l. Unbound ligands were removed at the end of incubation by rapid filtration on Whatman GF/C filters with a 1225 Sampling Manifold (Millipore). The filters were washed once with 15 ml of ice-cold assay buffer, and the filter-retained radioactivity was determined by liquid scintillation counting.
Immunocytochemistry-Transfected cells plated on poly-L-lysinecoated (25 g/ml) coverslips were fixed in 3% paraformaldehyde (in PBS) 24 h after transfection. When membrane permeabilization was required, cells were treated with PBS plus 0.1% (v/v) Triton X-100 for 10 min following fixation. Cells were washed three times in PBS and blocked for 10 min in PBS containing 10% fetal bovine serum and 0.5% bovine serum albumin. An anti-Myc antibody (Invitrogen), diluted 1:100, was applied for 1 h, followed by three washes in PBS. A fluorescein-conjugated anti-mouse IgG (Calbiochem), diluted 1:100, was applied for 1 h. Coverslips were washed three times in PBS, mounted on a glass slide, and examined using a fluorescent microscope (Zeiss).
Protein Analysis-Cells were transfected with tagged or untagged GABA A receptor subunit constructs and harvested after 48 h. Ni-NTAagarose Beads (Qiagen) were used to purify the His 6 -tagged proteins under native conditions. The purified His 6 proteins were eluted and separated on SDS-PAGE gels for immunoblotting using an anti-Myc (Invitrogen) or anti-v5 antibody (Invitrogen). Antibodies were diluted 1:5000.

Mutation of Ser-171 in ␥2 Prevents Benzodiazepine Binding to the GABA A Receptor-Serine-171 and tyrosine-172 in the ␥2
subunit of GABA A receptors are extremely conserved in the GABA A receptor subunit family and have recently been found to be crucial to the subunit association and cell surface localization of ␥2 (16). The present study further addresses the following two issues: (i) if one of the two conserved residues is more important than the other in mediating the subunit interaction and cell surface expression of ␥2; and (ii) if other amino acids that flank Ser-171/Tyr-172 are also required for the above function.
To answer these questions, we performed a single amino acid substitution at serine 171, tyrosine 172, and five other less conserved residues that flank Ser-171 and Tyr-172 (Fig. 1A). To examine the effect of mutations on receptor binding, individual ␥2 mutants were co-transfected with the ␣2 and ␤1 subunits into human embryo kidney 293 cells.  Fig. 1B), as compared with the binding from control cells co-expressing ␣2, ␤1, and the wild-type ␥2 subunit. Other amino acid substitutions, including the Y172F mutation within ␥2, showed no significant effect on the binding of both ligands.
Mutation of Ser-171 in ␥2 Prevents Its Cell Surface Localization-Binding of benzodiazepines to the GABA A receptor requires the presence of all three subunits (␣, ␤, and ␥) on the cell surface (11)(12)(13)(14)(15). Here, we examined the subcellular localization of the ␥2 mutants using immunofluorescence staining. We focused on the localization of the Ser-171 and Tyr-172 mutants, because these mutants exhibited a disruptive effect on ligand binding (see above). To facilitate protein detection, the ␥2 constructs were fused to a Myc-His tag at the 3Ј-terminus, and a second Myc tag was inserted between the ninth and tenth residues of the mature ␥2 peptide as described previously (16). Cells co-expressing ␣2, ␤1, and the Myc-tagged, wild-type ␥2 subunit ␥2 Myc showed robust cell surface staining, which was detected by the anti-Myc antibody ( Fig. 2A). However, no immunostaining was detected on the surface of the cells co-expressing ␣2, ␤1, and either a ␥2 S171G Myc (Fig. 2C) or a ␥2 S171C Myc mutant (Fig. 2E). When the cell membrane was permeabilized, all transfected cells showed a strong perinuclear staining typical of an endoplasmic reticulum-staining pattern (Fig. 2, B, D,  and F). Mutation of tyrosine 172 to phenylalanine showed no effect on the cell surface expression of ␥2 (Fig. 2I), whereas mutating the same residue to threonine slightly decreased the ␥2 cell surface-staining intensity (Fig. 2G).
Mutation of Ser-171 in ␥2 Inhibits Subunit Interaction-Heterologous interaction of GABA A receptor subunits takes place in the endoplasmic reticulum, where access to the cell surface is restricted only to the correctly assembled GABA A receptors (11)(12)(13)(14)(15). To study the interaction of the ␥2 mutants with other receptor subunits, a v5 tag was fused to the ␣2 and ␤1 subunits at the C terminus (16). The v5-tagged ␣2 and ␤1, and the Myc-His-tagged ␥2 or ␥2 mutants were co-expressed in 293 cells as detailed in Fig. 3. Subunit complexes were purified using Ni-NTA-agarose beads under native conditions and separated on SDS-PAGE gels. Immunoblots were probed with an anti-Myc and an anti-v5 antibody, respectively. Interaction of the ␣2 v5 and ␤1 v5 subunits with ␥2 Myc was demonstrated by the co-precipitation experiment (Fig. 3, lane 2). Similar subunit interaction was also detected among the ␣2 v5 and ␤1 v5 subunits and the ␥2 Y172F Myc mutant (Fig. 3, lane 6). An apparent decrease in subunit interaction was observed among the ␣2 v5 and ␤1 v5 subunits and the ␥2 Y172T Myc mutant (Fig. 3, lane 5). This decrease is consistent with the reduced receptor binding (Fig. 1) and reduced cell surface immunostaining of ␥2 (Fig. 2G). Subunit interactions of the ␣2 v5 and ␤1 v5 subunits with either the ␥2 S171G Myc or ␥2 S171C Myc mutant were drastically decreased (Fig. 3,  lanes 3 and 4), demonstrating a key role for serine 171 of ␥2 in mediating heterologous subunit interaction.
Ser-171 Is Conserved in the Ligand-gated Ion Channel Superfamily-Members of the ligand-gated ion channel receptors share significant amino acid sequence similarity and form a gene superfamily. Sequence alignment based on our BLAST data and the published results (1,18) shows that Ser-171 in ␥2 is highly conserved within the entire ligand-gated ion channel receptor superfamily, which includes ACh, GABA A , 5-hydroxytryptamine type 3, glycine, and glutamate receptors (Fig.  4). However, the conservation of Tyr-172 in ␥2 is more restricted to the members of the GABA A receptor family (16). The sequences shown in Fig. 4 are a subset of the alignment and cover the regions of ␤-strand 7 and 8 linked by loop 8 (18). The residues shaded in blue on Fig. 4 share a high degree of structural conservation (1). DISCUSSION The extracellular domains of the GABA A receptor subunits play essential roles in receptor assembly. For example, a ␥2 subunit containing only the extracellular domain can form a stable complex with the ␣1 or ␤3 subunits (12). Residues 58 -67 within the ␣ subunit are required in the assembly of the ␣␤ receptors, and Gln-67 within the ␣1 subunit has a major role in mediating its cell surface expression with the ␤3 subunit (19). Also, several N-terminal residues in the ␤ subunits direct their assembly with ␥ subunits (15,20).
The essential role of Ser-171/Tyr-172 in mediating the function of ␥2 was identified in the characterization of a naturally occurring ␥2 subunit splice variant, ␥2XL (16). ␥2XL contains an additional alternative exon that is inserted in the extracellular domain between Ser-171 and Tyr-172 of the mature ␥2 subunit. This variant neither co-precipitates with ␣2 and ␤1,  nor is it expressed on the cell surface. Such a disruptive effect is also found in a ␥2 mutant in which both Ser-171 and Tyr-172 are converted to glycine and threonine, respectively. Interestingly, Ser-171 and Tyr-172 are the residues that flank the inserted alternative exon and are also extremely conserved in the GABA A receptor subunit family members (16).
The findings described in this paper demonstrate that the mutation of ␥2 at serine 171, either to cysteine or glycine, disrupts the interaction of the ␥2 mutant with ␣2 and ␤1 in 293 cells. As a result, only ␣2 and ␤1 are oligomerized and expressed on the cell surface. The mutation of tyrosine 172 in ␥2 to phenylalanine or threonine results in distinct consequences. The tyrosine-to-phenylalanine mutation in ␥2 generates no disruptive effects, whereas the tyrosine-to-threonine mutation results in a partial decrease in both the subunit interaction and the cell surface expression of the ␥2 mutant when it is coexpressed with ␣2 and ␤1 in 293 cells. Unlike the Ser-171 mutants, the Y172T mutant also decreases the receptor binding of [ 3 H]muscimol, indicating that the tyrosine-to-threonine mutation in ␥2 may reduce access of the ␣2␤1 receptor to the cell surface. Tyrosine is most similar to phenylalanine but structurally distinct from threonine.
To map our mutagenesis data onto a three-dimensional context, we used a recent homology model of the extracellular part of the GABA A receptor (1) to predict the structure-function role of serine 171 and tyrosine 172. This homology model of ␥2 uses the solved three-dimensional structure of the acetylcholinebinding protein, a ligand-gated ion channel superfamily member, as a structural template (Protein Database identifier: 1I9b) (18). There is a 44% sequence similarity and a 25% sequence identity between AChBP and the ␥2 extracellular domain from amino acids 24 to 179. Although the homology between AChBP and ␥2 is relatively low, structure is known to be more conserved than sequence within protein families (1,21). Several other groups have recently used the AChBP homology model to predict the three-dimensional structure of the GABA A receptor subunits (22,23).
Based on this homology model, the extracellular domain of ␥2 is predicted to form a structure that is rich in ␤-sheets with an immunoglobulin-like topology. The conserved serine 171 in ␥2 is located at the end of ␤-strand 7, near the surface of a subunit interaction site (Figs. 4 and 5). Loop 8, which links the two ␤-strands (␤-strand 7 and 8), consists of two consecutive tight turn structures, each stabilized by hydrogen bonds. Residues preceding Ser-171 (glutamate 168, phenylalanine 169, and serine 170) are located in ␤-strand 7. Residues tyrosine 172, glycine 173, and proline 175 fall within loop 8. The side chain of Tyr-172 appears to be in direct contact with the ␤ subunit. The location of Ser-171 and the orientation of its side chain suggest that this serine may play a critical role in stabilizing the first turn (residues 171-174) by forming hydrogen bonds between its hydroxyl side-chain group and the mainchain amide group of Tyr-174 (Fig. 5C). As the side-chain of Ser-171 is pointing inward and is tightly packed, only limited changes of the side-chain volume appear possible without changing the conformation of the turn. Hence, mutations of Ser-171 in ␥2, which disrupt the hydrogen bonding or introduce a bulkier side chain that cannot be accommodated, might alter the conformation of the loop turn and lead to a direct or allosteric change in the subunit contact site(s). Serine and cysteine are stereochemically very similar, with nearly equivalent volumes. Prevention of the oligomerization of ␥2 with ␣2 and ␤1 by the serine-to-cysteine mutation at position 171 emphasizes the importance of forming the turn-stabilizing hydrogen bonds. It appears that even a subtle change at position 171 may be magnified through subsequent adjustments along the loop, con-siderably altering the subunit-subunit binding site. Mutations of serine 171 may therefore be more detrimental than mutations of tyrosine 172, as the former may cause more global changes in the positioning of the entire loop 8, whereas mutations of the latter only result in localized changes that may be better tolerated.
Finally, because serine 171 and tyrosine 172 are both extremely conserved among the members of the GABA A receptor subunit family (16), it would be interesting to speculate whether or not the same residues on other GABA A receptor subunits play similar roles in the assembly of GABA A receptor subunits. Additional studies of these conserved residues need to be performed to determine the structure-function relationships of other GABA A receptor subunits.