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Stoichiometry of a Ligand-gated Ion Channel Determined by Fluorescence Energy Transfer

1999; Elsevier BV; Volume: 274; Issue: 15 Linguagem: Inglês

10.1074/jbc.274.15.10100

1083-351X

Sophie J. Farrar, Paul J. Whiting, Timothy P. Bonnert, Ruth M. McKernan,

Neuroscience and Neuropharmacology Research

We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged α1-, β2-, or γ2-subunits of the hetero-oligomeric γ-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the α1(c-Myc)β2γ2 and the α1β2(c-Myc)γ2 receptors was twice that obtained with α1β2γ2(c-Myc), suggesting that there are 2× α-, 2× β-, and 1× γ-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining α1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with α1(c-Myc)β2γ2 and α1β2(c-Myc)γ2, but no significant energy transfer was observed with α1β2γ2(c-Myc) receptors, indicating the absence of a second γ-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, α1β2γ2, is composed of two copies each of the α- and β-subunits and one copy of the γ-subunit (i.e.(α1)2(β2)2(γ2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins. We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged α1-, β2-, or γ2-subunits of the hetero-oligomeric γ-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the α1(c-Myc)β2γ2 and the α1β2(c-Myc)γ2 receptors was twice that obtained with α1β2γ2(c-Myc), suggesting that there are 2× α-, 2× β-, and 1× γ-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining α1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with α1(c-Myc)β2γ2 and α1β2(c-Myc)γ2, but no significant energy transfer was observed with α1β2γ2(c-Myc) receptors, indicating the absence of a second γ-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, α1β2γ2, is composed of two copies each of the α- and β-subunits and one copy of the γ-subunit (i.e.(α1)2(β2)2(γ2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins. γ-aminobutyric acid, type A γ-aminobutyric acid monoclonal antibody europium cryptate minimum essential medium fetal calf serum Understanding the assembly and molecular interactions of proteins is a rapidly growing field in biology. Many techniques have been used to probe the interaction of proteins including, for example, immunoprecipitation, yeast two-hybrid analysis, circular dichroism, and more recently fluorescence energy transfer between two proteins in close proximity (1Mathis G. Clin. Chem. 1995; 41: 1391-1397Crossref PubMed Scopus (319) Google Scholar, 2Thomas D.D. Carlsen W.F. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5746-5750Crossref PubMed Scopus (158) Google Scholar, 3Damjanovich S. Bene L. Abdelkrim A. Goldman C.K. Sharrow S. Waldmann T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13134-13139Crossref PubMed Scopus (120) Google Scholar). We have used the technique of fluorescence resonance energy transfer to investigate the stoichiometric assembly of a GABAA1receptor, a member of the major inhibitory ligand-gated ion channel family in the brain. It is proposed that the GABAA receptors are hetero-oligomeric pentamers composed of α, β, and a third subunit type, γ, δ, or ε. This is based on an analogy with other ligand-gated ion channels such as the nicotinic acetylcholine receptor (4Raftery M.A. Hunkapiller M.W. Strader C.D. Hood L.E. Science. 1980; 208: 1454-1457Crossref PubMed Scopus (360) Google Scholar, 5Cooper C.E. Couturier S. Ballivet M. Nature. 1991; 350: 235-238Crossref PubMed Scopus (402) Google Scholar), their size as measured by gel filtration and sucrose density centrifugation (6Mamalaki C. Barnard E.A. Stephenson F.A. J. Neurochem. 1989; 52: 124-134Crossref PubMed Scopus (31) Google Scholar, 7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar), and their image under electron microscopy (8Nayeem N. Green T.P. Martin I.L. Barnard E.A. J. Neurochem. 1994; 62: 815-818Crossref PubMed Scopus (378) Google Scholar). To date, 15 GABAA subunits have been cloned (α1–6, β1–4, γ1–3, δ, and ε), and expression studies reveal that for the most characterized subtypes at least one α, one β, and one γ-subunit are required to recover a fully functional GABAA receptor (9Pritchett D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 338: 582-585Crossref PubMed Scopus (1150) Google Scholar). We have investigated the stoichiometry of the most abundant GABAAreceptor isoform in the brain, α1β2γ2, which is responsible for at least some of the therapeutic effects of benzodiazepines, barbiturates, and steroids (10McKernan R.M. Whiting P.J. Trends Neurosci. 1996; 19: 139-143Abstract Full Text PDF PubMed Scopus (1085) Google Scholar). Receptors modified with a c-Myc epitope on either the α1-, β2-, or γ2- subunit were expressed transiently in HEK293 cells. It has previously been shown that receptors transiently transfected in these cells using the same methods as employed here are appropriately assembled into αβγ-hetero-oligomers and are expressed on the cell surface. Other subunit assemblies do not reach the cell surface and are retained in the endoplasmic reticulum (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). mAbs to the c-Myc epitope were derivatized with the fluorescence donor, europium cryptate (EuK), or the fluorescent energy acceptor, XL-665. Europium, encaged by cryptate, emits a strong long lived fluorescent signal at 620 nm when illuminated with light at 337 nm from a nitrogen laser, which can be time-resolved from short lived background fluorescence. In addition to being used as a single label, analogous to a radiolabel for example, EuK also serves as an energy donor. The fluorescent signal produced by EuK can be transferred to an acceptor molecule if it is in close enough proximity. The recipient molecule for this fluorescent resonance energy transfer is a modified allophycocyanine, XL665, which fluoresces at 665 nm (for review of the homogeneous time-resolved fluorescence technology see Mathis (1Mathis G. Clin. Chem. 1995; 41: 1391-1397Crossref PubMed Scopus (319) Google Scholar)). The transfer of energy from europium cryptate-labeled c-Myc antibodies to XL-665-labeled antibodies is therefore indicative of the two antibodies being in close proximity. Because the energy transfer is 50% at a distance of 9.5 nm (1Mathis G. Clin. Chem. 1995; 41: 1391-1397Crossref PubMed Scopus (319) Google Scholar), this would require that the antibodies are very closely associated, for example in the same macromolecular complex. We used EuK-labeled c-Myc mAbs to quantify the number of subunits present on intact cells expressing α1β2γ2 subunits where each of the subunits was epitope-tagged with c-Myc and compared this with the number of receptors present using conventional radioligand binding. Fluorescence energy transfer was then used to confirm the stoichiometry of the receptor as (α1)2(β2)2(γ2)1. [methyl-3H]Ro15-1788 (87.0 Ci/mmol) was from NEN Life Science Products, Hertfordshire, United Kingdom. Flunitrazepam, GABA, and fetal calf serum were from Sigma. Minimal essential medium (MEM) was from Life Technologies, Inc. The GABAAreceptor α1-subunit was epitope-tagged by site-directed mutagenesis using methods described previously (13Wingrove P.B. Wafford K.A. Bain C. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4569-4573Crossref PubMed Scopus (216) Google Scholar). The epitope sequence (EQKLISEEDL) was introduced between Glu34and Leu35 (these two residues becoming the first and last amino acid of the epitope tag), just C-terminal to the putative signal peptide cleavage site. The β2 and γ2GABAA receptor subunits were epitope-tagged using a modified version of the pcDNA1Amp eukaryotic expression vector (pcDNA1AmpSignalMyc). This vector was constructed from the α1 c-Myc cDNA described above and contains the 5′-untranslated region of bovine GABAAα1-subunit (GenBank accession no. X05717), the signal peptide and six amino acids of the mature α1-subunit, and the c-Myc epitope tag sequence (EQKLISEEDL (14Evan G.I. Lewis G. Ramsay G. Bishop J.L. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2166) Google Scholar)) followed by a small polylinker into which the mature polypeptide of the subunit of interest can be inserted. The human β2-subunit c-Myc construct contains amino acids Glu38–Asn474 of β2, and the human γ2-subunit c-Myc construct contains amino acids Tyr46–le467. All constructs were confirmed by DNA sequencing. Constructs were prepared using polymerase chain reaction to generate the appropriate β2 and γ2 sequences, which were then subcloned into the pcDNA1AmpSignalMyc vector. DNAs were prepared for transfection by CsCl centrifugation. Transient transfection into HEK293 cells was performed exactly as described previously in detail using a 1:1:1 ratio of α1, β2, and γ2 cDNAs (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 15Wingrove P.B. Thompson S.A. Wafford K.A. Whiting P.J. Mol. Pharmacol. 1997; 52: 874-881Crossref PubMed Scopus (95) Google Scholar). The benzodiazepine site of the GABA receptor was labeled by the antagonist [3H]Ro15-1788, a radioligand frequently used to quantify receptors, because only fully assembled αβγ heterotrimers bind this ligand (7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar, 12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 16Duggan M.J. Pollard S. Stephenson F.A. J. Biol. Chem. 1991; 266: 24778-24784Abstract Full Text PDF PubMed Google Scholar). Nonspecific binding was determined using 10 μmflunitrazepam. Binding to the GABA binding site was carried out with [3H]muscimol, and 100 μm GABA was used to define nonspecific binding. Radioligand binding assays were carried out in a total volume of 0.5 ml in 10 mmKH2PO4, 100 mm KCl, pH 7.4, for 1 h at 4 °C prior to termination through Whatman GF/C filters followed by 3× 3-ml washes with cold assay buffer and scintillation counting. In binding studies carried out at one concentration of [3H]Ro15-1788, this was 1.8 nm. GABAA receptors solubilized from stably transfected cells were immunoprecipitated using anti-α1-, β-, or γ2-subunit antisera bound to protein A-Sepharose (7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar). 50 μl of polyclonal antiserum were incubated with 50 μl of packed protein A beads in a total volume of 1 ml of Tris-buffered saline for 1 h at room temperature. Receptors were solubilized from the cells using a deoxycholate/Triton buffer (1% Triton X-100, 0.5% deoxycholate, 0.1 mm KCl, 5 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 100 mm Tris-HCl, pH 8.2) by mixing cell membranes for 1 h at 4 °C with a detergent buffer at a protein concentration of 1 mg/ml. Aliquots (500 μl) of detergent-solubilized cell membranes were incubated with antibodies immobilized on protein A beads overnight at 4 °C. The receptor immobilized on protein A beads was washed three times with Tris-buffered saline/0.1% Tween 20 by centrifugation and resuspension and was finally resuspended in 0.5 ml of Tris-buffered saline, and 10–50-μl aliquots of packed beads were used for [3H]Ro15-1788 binding. The monoclonal anti-c-Myc mAb, 9-E10 (ATCC no. CRL-1729, Ref. 14Evan G.I. Lewis G. Ramsay G. Bishop J.L. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2166) Google Scholar) was used for these experiments. An antibody was purified from hybridoma supernatant by Cymbus Biotech, UK, and this was then derivatized with either the fluorophore, europium cryptate (at an average ratio of 9 molecules of europium cryptate per antibody molecule), or with the fluorescence acceptor, XL665 (at a ratio of 1 molecule of XL-665 per antibody molecule), by Cis-Bio International, Marcoule, France. Labeling of c-Myc mAb with EuK at this ratio gave 418 counts of fluorescence/s/fmol of antibody. Intact cells, transiently transfected with GABAA receptors with c-Myc-tagged subunits, were harvested by scraping, washed once by centrifugation (20 s at 1,000 rpm in a bench top Eppendorf Microcentrifuge), and resuspended in MEM + 5% FCS. [3H]Ro15-1788 binding (1.8 nm) was carried out to determine receptor density on intact cells. Cells containing the equivalent of 100 fmol of receptor were incubated with various concentrations of europium cryptate-derivatized mAb (0.3–30 nm) in MEM + 5% FCS at room temperature, in a volume of 1 ml for 1 h on a rotamix wheel. After 3× 1-ml washes in MEM + 5% FCS, the cells were resuspended in 190 μl of MEM + 5% FCS, and the fluorescence signal derived from europium cryptate-labeled c-Myc mAbs was quantified in a 96-well low fluorescence microplate on the Packard discovery fluorescence plate reader, following the addition of 10 μl of 1 m KF (final concentration, 50 mm KF). In preliminary experiments the inclusion of 5% FCS was found to improve the viability of the cells, and KF was included to stabilize europium cryptate and prevent oxidation of unliganded cryptate. XL665-derivatized mAbs were added in excess (10 nm) to each well, and the plate was read over a 24-h time period. Energy transfer was optimal after the receptor had been incubated with c-Myc-XL665 for 1 h, and the signal was stable for up to 18 h afterward. Each of the α1-, β2-, and γ2-subunits was engineered to express a c-Myc epitope tag at the N terminus, and cells expressing c-Myc-tagged α-, β-, or γ-subunits or untagged receptors were generated by transient transfection. GABAA receptors were measured using two ligands, [3H]muscimol, a radioligand for the GABA binding site, and [3H]Ro15-1788, a radioligand for the benzodiazepine binding site, which binds only to fully assembled receptors that contain an α-, β-, and γ-subunit (7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar, 9Pritchett D.B. Sontheimer H. Shivers B.D. Ymer S. Kettenmann H. Schofield P.R. Seeburg P.H. Nature. 1989; 338: 582-585Crossref PubMed Scopus (1150) Google Scholar). In preliminary experiments it was important to confirm that a homogeneous population of fully assembled receptors was being expressed in the cells. This was done in two ways. First the number of binding sites for [3H]muscimol and [3H]Ro15-1788 was compared. Most evidence supports the presence of two [3H]muscimol binding sites and one [3H]Ro15-1788 binding site in a GABA receptor monomer in receptors immunoprecipitated from rat brain (7Hadingham K.L. Harkness P.C. McKernan R.M. Quirk K. Le Bourdelles B. Horne A.L. Kemp J.A. Barnard E.A. Ragan C.I. Whiting P.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6378-6382Crossref PubMed Scopus (69) Google Scholar, 22Quirk K. Gillard N.P. Ragan C.I. Whiting P.J. McKernan R.M. Mol. Pharmacol. 1994; 45: 1061-1070PubMed Google Scholar). Therefore, if the majority of receptors is correctly assembled in cell lines, the ratio of the B max values for [3H]muscimol binding:[3H]Ro15-1788 binding should be 2:1. It is possible that receptors could be expressed that are composed of α- and β-subunits only, but these would not bind [3H]Ro15-1788, and the ratio would therefore be higher. Saturation analysis of [3H]muscimol and [3H]Ro15-1788 binding was carried out in cells expressing the untagged α1-, β2-, and γ2-subunits. Maximal binding of [3H]muscimol was 2067 ± 98 fmol/mg of protein with a K d of 5.6 ± 0.7 nm(n = 3). Maximal binding of [3H]Ro15-1788 was 1226 ± 21 fmol/mg of protein with a K d of 0.7 ± 0.05 nm (n = 3). The ratio of the GABA:benzodiazepine sites was 1.74 ± 0.11, which is in agreement with the expression of αβγ heterotrimeric receptors and no significant expression of receptors that contain only αβ-subunits. Second, the ability of antibodies raised against the α1-, β2-, and γ2-subunit to immunoprecipitate all the [3H]Ro15-1788 binding sites from a solubilized cell membrane preparation was compared. The majority of receptors could be immunoprecipitated with antibodies raised against the α1-, β-, or γ2-subunits (α1, 74 ± 3%; β, 81 ± 7%; and γ2, 90 ± 4% of [3H]Ro15-1788 binding sites, n = 3). This confirms that the receptors that bind [3H]Ro15-1788 contain at least one α-, one β-, and one γ-subunit. The number of c-Myc-tagged subunits was quantified by labeling with anti-c-Myc mAb derivatized with europium cryptate, as illustrated in Fig. 1 (using the subunit arrangement proposed by Tretter et al. (11Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar) as a model). The presence of a c-Myc tag on the N terminus of the GABAAreceptor subunits did not affect the expression of the receptor. The number of binding sites present on cells was unaffected by the addition of the epitope tag, and saturation analysis of [3H]Ro15-1788 binding showed that there was no major difference in the affinity of receptors for ligand (K d is as follows: α1β2γ2 = 1.3 nm; α1(c-Myc)β2γ2 = 1.1 nm; α1β2(c-Myc)γ2= 1.4 nm; α1β2γ2(c-Myc) = 1.3 nm, n = 1). The density of receptors was unaffected by the expression of subunits with a c-Myc epitope tag, being 1.2–3.1 pmol/mg of protein. This is in agreement with previous studies where GABAA receptors were tagged with c-Myc and FLAG epitopes without compromising binding, function, or modulation of the GABAA receptor (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Preliminary experiments were carried out to determine the optimal conditions for the binding of EuK-labeled c-Myc mAbs. As shown in Fig. 2 B, the fluorescence signal emitted by EuK c-Myc at 620 nm was linear with receptor concentration. Therefore the binding of EuK-labeled c-Myc to the receptor was linear up to at least 100 fmol of receptor/ml. Background fluorescence from the plate and other reagents accounted for the fluorescence in the absence of added EuK, and this was typically 800–1500 counts/s as shown in Fig. 2 A. Binding of EuK c-Myc to cells expressing untagged receptors was significant (Fig.2 A). Efforts were made to try to reduce this nonreceptor-mediated binding by extensive washing following incubation with EuK-c-Myc mAb and preincubation of cells with IgG to block any IgG binding sites on the cells. Neither of these measures reduced nonspecific binding to control cells by more than 10% and were not routinely adopted. Instead, each experiment contained a wild type control (i.e. receptors expressed without any c-Myc epitope present), and nonspecific binding to these cells was subtracted from the total fluorescence signal, which was analogous to the methodology employed for defining nonspecific binding in a radioligand binding assay. Saturation binding experiments were carried out using control cells expressing α1β2γ2 and cells expressing receptors where either the α-, β-, or γ-subunit was epitope-tagged with c-Myc. Nonspecific binding of EuK c-Myc to control cells expressing an untagged receptor was subtracted from total binding at each concentration of antibody and was routinely 50% at 3 nm antibody. In all cases binding was saturable and had a high affinity, and EuK-labeled c-Myc bound to more sites in the α1(c-Myc)β2γ2 and α1β2(c-Myc)γ2 compared with α1β2γ2(c-Myc) as exemplified in Fig. 3. To quantitate this, the experiment was repeated on six separate occasions using 3 nm antibody, and the specific fluorescence counts are shown in Fig. 4 A. The ratio of fluorescence labeling to α1(c-Myc)β2γ2, α1β2(c-Myc)γ2, and α1β2γ2(c-Myc) was compared as shown in Fig. 4 B. This clearly shows that there are twice as many EuK c-Myc sites on receptors where the α- or β-subunit is tagged compared with receptors where the γ-subunit is tagged (ratio of α1(c-Myc)β2γ2:α1β2(c-Myc)γ2:α1β2γ2(c-Myc)= 1.89 ± 0.12:1.94 ± 0.18:1).Figure 4Fluorescence signal observed at 620 and 665 nm from cells expressing α1(c-Myc)β2γ2, α1β2(c-Myc)γ2, and α1β2γ2(c-Myc).Data shown are the mean ± S.E. of six independent experiments. The specific fluorescence counts observed at 620 nm for α1(c-Myc)β2γ2 were 315 ± 37/fmol and for α1β2(c-Myc)γ2 were 286 ± 41/fmol. These were both significantly higher than that observed with α1β2γ2(c-Myc), 156 ± 21/fmol. The mean ± S.E. and the ratio for binding relative to α1β2γ2(c-Myc) were calculated for each experiment and are shown in panel B. The ratios of binding for α1(c-Myc) β2γ2:α1β2γ2(c-Myc) and α1β2(c-Myc)γ2:α1β2γ2(c-Myc) were not significantly different from 2.0 (p < 0.05, Student's t test). Panel C, in the fluorescence energy transfer experiments, where emission was measured at 665 nm, there was no significant difference between the fluorescence counts produced by α1(c-Myc)β2γ2(16.7 ± 1.5) and α1β2(c-Myc)γ2 (17.8 ± 1.4), but both were significantly more than α1βγ2(c-Myc) (0.38 ± 2.4), which was not significantly more than 0.View Large Image Figure ViewerDownload (PPT) A comparison of the B max values for [3H]Ro15-1788 and EuK-c-Myc fluorescence can be used to calculate the number of binding sites for the antibody/benzodiazepine binding site. For α1(c-Myc)β2γ2 there are 315 ± 37 fluorescence counts/fmol of Ro15-1788 binding sites using 3 nm antibody and 1.8 nm[3H]Ro15-1788 (Fig. 4). Using Clarke's equation (occupancy = [ligand]/[ligand] + K i) this is equivalent to 610 ± 71 fluorescence counts/fmol of Ro15-1788 binding sites. Given that there are 418 fluorescence counts of EuK/fmol of antibody (see "Experimental Procedures"), there are calculated to be 1.46 ± 0.16 antibody binding sites/[3H]Ro15-1788 binding site. The limitations and multiple sources of error in this experiment (e.g.aggregation of antibody, heterogeneity of antibody labeling, etc.) would be more likely to lead to an underestimate of the number of antibody binding sites therefore the observation that there is more than one antibody binding site/[3H]Ro15-1788 binding site supports the most likely stoichiometry of 2 α-subunits/receptor:1 benzodiazepine site. The affinity of the EuK-derivatized c-Myc antibodies for the GABAA subunits was 4–6 nm as shown in Fig. 3. A concentration of 3 nm was selected for the fluorescence transfer studies because this would less than half-maximally saturate the receptor. Following incubation with 3 nm EuK-derivatized c-Myc mAb, cells were washed and incubated with excess XL665-derivatized c-Myc mAb, and fluorescence at 665 nm was measured in response to laser excitation. As shown in Fig. 4 C, a specific signal is observed, indicating energy transfer when either the α1- or the β2-subunit is epitope-tagged (16.7 ± 1.5 counts/fmol and 17.8 ± 1.4 counts/fmol) but not when the γ2-subunit is epitope-tagged. Because energy transfer can only take place when there are two c-Myc epitopes in close proximity,i.e. two subunits in the same receptor complex, this confirms that the stoichiometry of the GABAA receptor under study is (α1)2(β2)2(γ2)1. Furthermore, the lack of any observable signal with α1β2γ2(c-Myc) could be interpreted as evidence that receptors are not primarily expressed as clusters on the cell surface. If this were the case, it may be possible to observe energy transfer between γ2-subunits on separate receptors. To date, the stoichiometry of the GABAA receptor has been approached both directly and indirectly. Many laboratories have observed that a receptor can contain two different types of α-subunits (16Duggan M.J. Pollard S. Stephenson F.A. J. Biol. Chem. 1991; 266: 24778-24784Abstract Full Text PDF PubMed Google Scholar, 17Luddens H. Killisch I. Seeburg P.H. J. Recept. Res. 1991; 11: 523-551Crossref Scopus (49) Google Scholar, 18Zezula J. Sieghart W. FEBS Lett. 1991; 284: 15-18Crossref PubMed Scopus (33) Google Scholar, 19Mertens S. Benke D. Mohler H. J. Biol. Chem. 1993; 268: 5965-5973Abstract Full Text PDF PubMed Google Scholar, 20McKernan R.M. Quirk K. Prince R. Cox P.A. Gillard N.P. Ragan C.I. Whiting P. Neuron. 1991; 7: 667-676Abstract Full Text PDF PubMed Scopus (178) Google Scholar). Conflicting evidence has been obtained on whether two types of γ-subunit can coexist in a single receptor monomer with two studies proposing that two γ-subunits can be present (21Khan Z.U. Gutierrez A. De Blas A.L. J. Neurochem. 1994; 63: 1466-1476Crossref PubMed Scopus (60) Google Scholar, 22Quirk K. Gillard N.P. Ragan C.I. Whiting P.J. McKernan R.M. Mol. Pharmacol. 1994; 45: 1061-1070PubMed Google Scholar). There have been fewer studies on the presence of two β-subunits because the structural similarity of the β-subunits has precluded the development of antibodies, which clearly distinguish between them. There have been four studies that directly analyzed the stoichiometry of the receptor. The first two, chronologically, have taken an electrophysiological approach. Backus et al. (23Backus K.H. Arigoni M. Drescher U. Scheurer L. Malherbe P. Mohler H. Benson J.A. Neuroreport. 1993; 5: 285-288Crossref PubMed Scopus (118) Google Scholar) favor the composition 2× α, 1× β, and 2× γ based on measuring the outward current induced by point mutation of charged amino acids on either side of the TM2 domain. Chang et al. (24Chang Y. Wang R. Barot S. Weiss D.S. J. Neurosci. 1996; 16: 5415-5424Crossref PubMed Google Scholar) used a similar method (mutation of a lysine in TM2) to increase the sensitivity of the receptor to GABA in proportion to the number of mutant subunits present. In contrast to Backus et al. (23Backus K.H. Arigoni M. Drescher U. Scheurer L. Malherbe P. Mohler H. Benson J.A. Neuroreport. 1993; 5: 285-288Crossref PubMed Scopus (118) Google Scholar), they conclude that the stoichiometry is 2× α, 2× β, and 1× γ, as do Im et al. (25Im W.B. Prezenger J.F. Binder J.A. Dillon G.H. Alberts G.L. J. Biol. Chem. 1995; 270: 26063-26066Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) from studies of tandem subunit constructs. The most recent study used antibody labeling of chimeric subunits to determine the ratio of subunits present from Western blots, and they conclude that the structure is also 2× α, 2× β, and 1× γ (11Tretter V. Ehya N. Fuchs K. Sieghart W. J. Neurosci. 1997; 17: 2728-2737Crossref PubMed Google Scholar). The approach taken here has used fluorescently derivatized mAbs to quantify subunits relative to the benzodiazepine binding site on the receptor and fluorescent resonance energy transfer to confirm that the stoichiometry of the receptor is (α1)2(β2)2γ2. This method has several advantages over other previously described methods. 1) It uses intact cells; therefore only receptors that are expressed on the surface (and are therefore presumed to be correctly assembled) are considered (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 26Angelotti T.P. Macdonald R.L. J. Neurosci. 1993; 13: 1429-1440Crossref PubMed Google Scholar). 2) It involves minimal disruption of receptor structure by requiring only epitope tagging, which does not affect the expression or function of the receptor (12Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). 3) Only small amounts of transiently transfected material are required. Furthermore this approach has general applicability to other multisubunit cell surface proteins and can be used particularly to investigate the stoichiometry of subunits in GABAAreceptors composed of rarer subunits and in other ligand-gated ion channels. We thank Drs. Eric Trinquet and Gerard Mathis of Cis-Bio International for constructive discussion, Dr. Mark Shearman for contributing to the assay development, and Peter Wingrove for providing the transfected cells.