MolPharm xPharm- The Comprehensive Pharmacology Reference

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sur, C.
Right arrow Articles by McKernan, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sur, C.
Right arrow Articles by McKernan, R.

Vol. 54, Issue 5, 928-933, November 1998

Rat and Human Hippocampal alpha 5 Subunit-Containing gamma -Aminobutyric AcidA Receptors Have alpha 5beta 3gamma 2 Pharmacological Characteristics

Cyrille Sur, Kathleen Quirk, Deborah Dewar, John Atack, and Ruth McKernan

Department of Biochemistry, Merck Sharp and Dohme Research Laboratories, Harlow, Essex, CM20 2QR, UK (C.S., K.Q., J.A., R.M.) and Wellcome Surgical Institute, University of Glasgow, Glasgow, UK (D.D.)

    Summary
Top
Summary
Introduction
Procedures
Results
Discussion
References

The gamma -aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha 5beta 3gamma 2 and alpha 5beta 3gamma 3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha 5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha 5beta 3gamma 2 receptors (Kd = 1.7 ± 0.4 nM), compared with alpha 5beta 3gamma 3 receptors (Kd = 15 ± 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 ± 0.6 and 1.0 ± 0.2 nM, respectively, comparable to the affinity of alpha 5beta 3gamma 2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha 5beta 3gamma 2 but not alpha 5beta 3gamma 3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma 2-selective antibody yielded 19 ± 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma 3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha 5 and anti-gamma 2 or anti-alpha 5 and anti-gamma 3 antibodies established the preferential expression of alpha 5gamma 2 receptors, accounting for 22 ± 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha 5beta 3gamma 2 GABAA receptors in rat hippocampus, despite the clustering of alpha 5 and gamma 3 loci on the same chromosome.

    Introduction
Top
Summary
Introduction
Procedures
Results
Discussion
References

The GABAA receptor is the main inhibitory ligand-gated ion channel in the central nervous system. It contains modulatory sites for endogenous molecules such as the neurosteroids, as well as for many therapeutic drugs, such as barbiturates, anesthetics, and benzodiazepines (Sieghart, 1995). It is now generally accepted that the GABAA receptor is a pentameric protein with an integral chloride ion channel formed by the second transmembrane domain of each of the five subunits. A family of GABAA receptor subunits (alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, delta , and epsilon ) have been identified in mammalian brain using molecular cloning techniques (for review, see McKernan and Whiting, 1996; Davies et al., 1997; Whiting et al., 1997). At least one alpha  subunit, one beta  subunit, and one gamma  subunit are required to form fully functional receptors in vivo (Pritchett et al., 1989), and the combination of alpha  and gamma  subunits is a crucial determinant of the properties of the benzodiazepine binding site (Hadingham et al., 1993; Wafford et al., 1993; Luddens et al., 1994; Benke et al., 1996). The alpha 1 subunit-containing receptors exhibit BZ1-type pharmacological characteristics, characterized by a high affinity for zolpidem, whereas alpha 2, alpha 3, and alpha 5 subunits are present in BZ2-type receptors, which have a low affinity for zolpidem (Pritchett et al., 1989; Pritchett and Seeburg, 1990). A third class of GABAA receptors also exists; these receptors contain an alpha 4 or alpha 6 subunit and have a low affinity for most of the classical benzodiazepines (Luddens et al., 1990; Wisden et al., 1991; Wafford et al., 1996; Benke et al., 1997).

Receptors expressing an alpha 5 subunit together with beta  and gamma 2 subunits in cell lines are distinguished from BZ1 receptors by their low affinity for zolpidem (Pritchett and Seeburg, 1990; Luddens et al., 1994) and from other BZ2 receptors by their 10-20-fold higher affinities for Ro15-4513 (Hadingham et al., 1993; Luddens et al., 1994) and for several 8-substituted benzodiazepines (Gillard et al., 1994). Thus, alpha 5-containing receptors have a unique pharmacological profile.

In the rat central nervous system, alpha 5 subunit-containing receptors have restricted and well defined expression. In situ hybridization and immunocytochemistry studies have shown that this subtype is present in abundance in the CA1 and CA3 fields of the hippocampus, is present to a lesser extent in the cortex and olfactory bulb, and is virtually absent in other regions of the brain (Wisden et al., 1992; Fritschy and Mohler, 1995; Quirk et al., 1996).

The exact subunit composition of receptors containing an alpha 5 subunit in vivo is not known. The colocalization of alpha 5, beta 3, and gamma 3 subunits on chromosome 15 suggests possible associations among these subunits, and deletion of this locus reduces zolpidem-insensitive, radiolabeled benzodiazepine binding (Nakatsu et al., 1993). On the other hand, the pharmacological characteristics of alpha 5-containing receptors immunoprecipitated from rat brain are closer to those demonstrated in cells transfected with alpha 5beta 3gamma 2 than those observed with alpha 5beta 3gamma 3 (McKernan et al., 1991; Luddens et al., 1994). Similarly, electrophysiological studies indicated alpha 5beta 3gamma 2L as the isoform expressed by hippocampal CA1 pyramidal cells (Burgard et al., 1996).

Using a 50-100-fold selective ligand for alpha 5 subunit-containing receptors (Quirk et al., 1996), [3H]L-655,708, we describe here the pharmacological characteristics of alpha 5 subunit-containing receptors in rat and human brain and compare them with those of stable cell lines expressing alpha 5beta 3gamma 2 and alpha 5beta 3gamma 3 receptors. Implications for the structure of native alpha 5 subunit-containing receptors are discussed.

    Experimental Procedures
Top
Summary
Introduction
Procedures
Results
Discussion
References

Materials. [3H]Ro15-1788 and [3H]muscimol were purchased from New England Nuclear-Du Pont (Hertfordshire, UK). [3H]L-655,708 (76.7 Ci/mmol) was prepared as previously described (Quirk et al., 1996). CL218872 and CGS8216 were gifts from Lederle and Ciba-Geigy/Novartis, respectively, and other benzodiazepine site ligands were from Sigma Biochemicals or Research Biologicals Inc.

Human tissues. Hippocampi were from adult cadaveric brain tissue obtained from subjects without any neurodegenerative disease and without obvious morphological abnormalities of the hippocampi.

Membrane preparation and binding assays. P2 membranes were prepared from brain regions of adult male rats or from cadaveric hippocampi from adult human subjects as previously described (McKernan et al., 1991). Radioligand binding assays were performed with brain membranes or membranes prepared from stably transfected cell lines (Hadingham et al., 1993; Sur et al., 1997), with [3H]L-655,708 (0.1-40 nM), in a final volume of 0.5 ml containing 50-100 µg of protein in 10 mM Tris·HCl, 1 mM EDTA, pH 7.4, at 4°. For saturation analyses with rat and human hippocampi, 1 µM zolpidem was added to the assays, to prevent binding to other alpha  subunits at high [3H]L-655,708 concentrations. Similarly, immunoprecipitated receptor-protein A complexes were incubated with [3H]L-655,708 (20-24 nM), [3H]Ro15-1788 (20 nM), or [3H]muscimol (40 nM) for 1-2 hr at 4°. Nonspecific binding was defined with 10 µM flunitrazepam or 100 µM GABA, for 3H-benzodiazepine or [3H]muscimol binding, respectively. After 1-2-hr incubations at 4°, assay mixtures were filtered through Whatman GF/B filters using a cell harvester (Brandel) and were washed four times with cold buffer. Filters were immersed overnight in scintillation cocktail, and radioactivity was determined in a Beckman liquid scintillation counter. Data points were fitted by nonlinear regression analysis (Excel; Microsoft); for competition experiments, the Ki values were calculated according to the Cheng-Prusoff equation (Cheng and Prusoff, 1973).

Immunoprecipitation of GABAA receptors. The antibodies used in this study were previously characterized and shown to be subunit specific (McKernan et al., 1991; Quirk et al., 1994a, 1994b, 1995). Immunoprecipitation of receptors was carried out using antibodies to GABAA receptors, as previously described (McKernan et al., 1991; Quirk et al., 1994a). Briefly, 100 µl of protein A-Sepharose beads was incubated with 40-80 µl of antibody for 1 hr at room temperature. After washing with Tris-buffered saline (10 mM Tris·HCl, 150 mM NaCl) containing 0.1% Tween 20, beads were incubated overnight at 4° with deoxycholate (0.5%)-solubilized receptors from hippocampus. Beads were washed twice with Tris-buffered saline/Tween 20, and binding studies were performed using 25-50 µl of packed beads in each tube. Parallel experiments with an antibody directed against the 5-hydroxytryptamine3 receptor served as controls for the immunoprecipitation experiments.

    Results
Top
Summary
Introduction
Procedures
Results
Discussion
References

[3H]L-655,708 binding characteristics. To determine the affinity of [3H]L-655,708, saturation experiments were performed with membranes prepared from alpha 5beta 3gamma 2- or alpha 5beta 3gamma 3-expressing cells, as well as rat and human hippocampi (Fig. 1 and Table 1). They revealed a 9-fold selectivity of [3H]L-655,708 for alpha 5beta 3gamma 2 versus alpha 5beta 3gamma 3 receptors, with Kd values of 1.7 ± 0.4 and 15 ± 3 nM, respectively. Saturation analysis of [3H]L-655,708 binding to rat and human hippocampi revealed the existence of a single high affinity binding site, with Kd values of 2.2 ± 0.6 and 1.1 ± 0.2 nM, respectively. These experiments also showed no difference (p > 0.46, t test) in the numbers of [3H]L-655,708 binding sites in human (340 ± 184 fmol/mg of protein, mean ± standard deviation, five experiments) and rat (251 ± 74 fmol/mg of protein, mean ± standard deviation, three experiments) hippocampi. Determination of the [3H]L-655,708 (2 nM)/[3H]Ro15-1788 (1.8 nM) ratio, however, indicated a slightly higher proportion of alpha 5-containing receptors in human hippocampus, with a ratio of 0.28 ± 0.04 (mean ± standard error, two experiments), compared with a ratio of 0.15 ± 0.02 (mean ± standard error, six experiments) for rat hippocampus (p < 0.005, t test).


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 1.   Saturable binding of [3H]L-655,708 to human recombinant alpha 5beta 3gamma 2 and alpha 5beta 3gamma 3 GABAA receptors and alpha 5-containing receptors from rat and human hippocampal membranes. The linear Scatchard plots (insets) show that [3H]L-655,708 binds to a single population of sites. Data shown are from a representative experiment that was performed at least three times. The binding parameters for these experiments were as follows: alpha 5beta 3gamma 2, Kd = 2.4 nM, Bmax = 223 fmol/mg of protein; alpha 5beta 3gamma 3, Kd = 10.2 nM, Bmax = 186 fmol/mg of protein; rat hippocampal membranes, Kd = 1.7 nM, Bmax = 170 fmol/mg of protein; human hippocampal membranes, Kd = 0.92 nM, Bmax = 200 fmol/mg of protein.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1
Affinities of [3H]L-655,708 for alpha 5-containing receptors

Data shown are the mean ± standard error of three to five experiments performed in triplicate.

Evidence that rat and human alpha 5-containing receptors display alpha 5beta 3gamma 2 pharmacological characteristics. Competition experiments using [3H]L-655,708 (2-3 nM) and seven representative benzodiazepine site ligands from different chemical series were carried out in cell lines and hippocampal membranes. The results (Table 2) established the selectivity of some compounds for either alpha 5beta 3gamma 2 (CGS8216, L-655,708, and diazepam) or alpha 5beta 3gamma 3 (CL218872) receptors. More specifically, CGS8216 demonstrated 14-fold selectivity for alpha 5beta 3gamma 2, whereas CL218872 exhibited 5-fold selectivity for alpha 5beta 3gamma 3 receptors. Correlation plots demonstrated a good relationship between the affinities of these compounds for alpha 5beta 3gamma 2 receptor-expressing cells and rat or human [3H]L-655,708 binding sites (Fig. 2, A and B). Furthermore, there was an excellent correlation of the pharmacological characteristics of rat and human alpha 5 receptors (Fig. 2C). In contrast, a nonsignificant correlation (p > 0.05, Spearman correlation) was observed for the pharmacological characteristics of hippocampal [3H]L-655,708 binding sites and the alpha 5beta 3gamma 3 isoform (data not shown).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 2
Affinities of benzodiazepine site ligands for alpha 5-containing receptors

Ki values determined with [3H]L-655,708 (2-4 nM) are the mean ± standard error of two to eight determinations performed in triplicate. The Hill coefficients were not different from unity except for CL218872 in rat and human hippocampi, where values of 0.64 ± 0.08 and 0.63 ± 0.07, respectively, were determined. The numbers before the drug names are used in Fig. 2.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 2.   Logarithmic-logarithmic plots of Ki values (nM) (mean ± standard error) for seven benzodiazepine site ligands, showing the correlation (Spearman correlation) between the pharmacological characteristics of rat (A) (r2 = 1) and human (B) (r2 > 0.92) hippocampal alpha 5-containing receptors and the alpha 5beta 3gamma 2 isoform, as well as between rat and human hippocampal alpha 5 receptors (C) (r2 > 0.92). Numbers near symbols, compounds listed in Table 2.

Pharmacological characteristics of immunoprecipitated rat hippocampal GABAA receptors. As shown in Fig. 3, solubilized and immunoprecipitated alpha 5-containing GABAA receptors retained high affinity (Kd = 3.7 ± 1.3 nM) [3H]L-655,708 binding. To gain more insight into the structure of rat hippocampal alpha 5-containing receptors, immunoprecipitation experiments were performed with several subunit-specific antibodies, and the binding of [3H]L-655,708 (20-24 nM) and [3H]Ro15-1788 (20 nM) was determined (Table 3). Because of the high concentration of [3H]L-655,708 used, binding was determined in the presence of zolpidem (1 µM), to prevent [3H]L-655,708 binding with low affinity to alpha 1, alpha 2, and alpha 3 subunits. Because we anticipated the lack of 3H-benzodiazepine binding to receptors precipitated with antibodies to gamma 1 or delta  subunits (Ymer et al., 1990; Quirk et al., 1995), the binding of [3H]muscimol (which binds to the GABA binding site of GABAA receptors) was measured in parallel experiments, to confirm that immunoprecipitation had occurred. All benzodiazepine sites (95 ± 6%) immunoprecipitated with anti-alpha 5 antibody, as determined with [3H]Ro15-1788, exhibited [3H]L-655,708 binding. Antibodies selective for alpha 1, alpha 2, or alpha 3 subunits were able to precipitate 3, 6, and 7%, respectively, of [3H]Ro15-1788 binding sites that also bound [3H]L-655,708 (Table 3). These populations of receptors accounted for a small proportion of total GABAA receptors.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 3.   Saturation analysis of [3H]L-655,708 binding to anti-alpha 5-immunoprecipitated receptors from rat hippocampus. The linear Scatchard plot (inset) indicates saturable binding of the radioligand to a single class of sites.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 3
Percentage of [3H]L-655,708/[3H]Ro15-1788 binding in immunoprecipitated receptors from rat hippocampus

Data shown are the mean ± standard error of two to five experiments.

Among the three gamma  subunits assayed, only antibodies to gamma 2 immunoprecipitated [3H]L-655,708 binding sites. These alpha 5gamma 2 subunit-containing receptors represented 19 ± 4% (mean ± standard error, five experiments) of total [3H]Ro15-1788 binding sites. The anti-gamma 3 antibody immunoprecipitated some [3H]Ro15-1788 binding sites (5.4 ± 2.4%, two experiments) but no [3H]L-655,708 binding, whereas no benzodiazepine binding was observed after precipitation with anti-gamma 1 or anti-delta antibodies.

Additional evidence for the presence of native alpha 5beta 3gamma 2 receptors in rat hippocampus was provided by additive immunoprecipitation experiments with anti-alpha 5, -gamma 2, and -gamma 3 antibodies, using [3H]muscimol binding (Fig. 4). Solubilized GABAA receptors from hippocampus were immunoprecipitated either with a single antibody or with alpha 5gamma 2 or alpha 5gamma 3 combinations. As shown in Fig. 4, anti-gamma 2 antiserum immunoprecipitated the larger amount of [3H]muscimol binding sites (59%), whereas anti-alpha 5 and anti-gamma 3 precipitated 31 and 7.6%, respectively. Interestingly, the antibody pair for alpha 5gamma 2 immunoprecipitated 66% of [3H]muscimol binding sites, a proportion much smaller than the sum of anti-alpha 5- and anti-gamma 2-precipitated sites (31 + 59 = 90%). In contrast, the quantity of [3H]muscimol binding sites immunoprecipitated by the pair for alpha 5gamma 3 (38%) corresponds to the sum of individually precipitated receptors (31 + 7.6 = 38.6%). Results from another experiment yielded similar values for immunoprecipitated [3H]muscimol binding sites with antibodies for alpha 5 (37%), gamma 2 (91%), gamma 3 (11%), alpha 5gamma 2 (109%), and alpha 5gamma 3 (49%). The difference between the calculated and measured values for the alpha 5gamma 2 tandem indicates that this subunit combination accounts for 21.5 ± 2.5% (mean ± standard error, two experiments) of [3H]muscimol binding sites in rat hippocampus.


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 4.   Immunoprecipitation of GABAA receptor binding sites from rat hippocampus by anti-alpha 5, -gamma 2, and -gamma 3 antibodies. Binding of [3H]muscimol (40 nM) was measured with solubilized receptors precipitated by either a single antibody or a combination of antibodies, as indicated. The calculated values for pairs of antibodies represent the sums of receptors immunoprecipitated by each antibody alone. In this representative experiment, the following values were determined: alpha 5, 31.4%; gamma 2, 59.4%; gamma 3, 7.6%; alpha 5gamma 2, 66.7%; alpha 5gamma 3, 38.1% of [3H]muscimol binding sites.

    Discussion
Top
Summary
Introduction
Procedures
Results
Discussion
References

Pharmacological evidence for alpha 5beta 3gamma 2 as a native GABAA receptor isoform. In a previous study, it was shown that L-655,708 has at least a 50-fold selectivity for alpha 5 versus alpha 1, alpha 2, alpha 3, and alpha 6 subunit-containing receptors and that the tritiated compound [3H]L-655,708 binds rapidly and reversibly to brain membranes, establishing this drug as a specific tool to investigate native alpha 5-containing receptors in more detail (Quirk et al., 1996). Here, the pharmacological characteristics of alpha 5 subunit-containing receptors from rat and human hippocampus have been analyzed with the selective tritiated ligand L-655,708 and compared with those of recombinant human alpha 5beta 3gamma 2 or alpha 5beta 3gamma 3 GABAA receptors expressed in cell lines.

Scatchard analysis of [3H]L-655,708 binding showed that alpha 5 receptors have similar levels of expression in human and rat hippocampus, as indicated by comparable Bmax values. The rat Bmax value (251 fmol/mg of protein) is similar to that measured in a previous study (Quirk et al., 1996) and accounts for 15% of [3H]Ro15-1788 binding sites, in agreement with results from immunoprecipitation experiments (McKernan et al., 1991; Mertens et al., 1993). In human hippocampus, alpha 5 receptors seem to be more abundant, representing 28% of [3H]Ro15-1788 binding sites. Although this value was not determined from saturation analysis, the observed difference is not accounted for by reduced Ro15-1788 affinity for human alpha 5 receptors (Table 2). However, we noticed an important variation in the [3H]L-655,708 Bmax values (coefficient of variation = 0.54), and a larger number of human hippocampal specimens should be investigated to confirm this apparent higher proportion of alpha 5-containing receptors. In addition, the saturation experiment data showed that [3H]L-655,708 has some binding selectivity (9-fold) for alpha 5beta 3gamma 2 receptors and shows similar Kd values for this isoform and native receptors in rat and human hippocampus.

The similarity of the pharmacological characteristics of alpha 5beta 3gamma 2 and hippocampal alpha 5-containing receptors was further established using several selective compounds for alpha 5beta 3gamma 2 receptors (CGS8216, diazepam, L-655,708, and flunitrazepam) or alpha 5beta 3gamma 3 receptors (CL218872) (Luddens et al., 1994; Hadingham et al., 1995). The rank order of the tested compounds matches and extends the reported data for native (McKernan et al., 1991; Quirk et al., 1996) and recombinant (Pritchett and Seeburg, 1990; Luddens et al., 1994) alpha 5-containing receptors and indicates that alpha 5beta 3gamma 2 is the major isoform of alpha 5-containing receptors expressed in rat and human hippocampus. These data are also in agreement with results from electrophysiological recordings of cells expressing alpha 5beta 3gamma 2L receptors and hippocampal CA1 pyramidal neurons, indicating that this isoform is a native GABAA receptor (Burgard et al., 1996).

Structure of rat hippocampal alpha 5 subunit-containing receptors. The quantitative immunoprecipitation results clearly indicated that our specific anti-gamma 3 antibody (Quirk et al., 1994a) did not precipitate any [3H]L-655,708 binding sites from rat hippocampus but precipitated >5% of [3H]Ro15-1788 binding sites. This value is similar (p > 0.31, t test) to the 9.3 ± 1.7% of [3H]muscimol binding sites immunoprecipitated from rat hippocampus and fits with the low level of gamma 3 subunit expression in rat hippocampus (Herb et al., 1992; Wisden et al., 1992). Furthermore, additive immunoprecipitation experiments similar to those used to demonstrate gamma 2gamma 3 coassembly (Quirk et al., 1994a) failed to support an association of gamma 3 with the alpha 5 subunit. In contrast, measured percentages for the alpha 5gamma 2 combination correspond almost exactly to theoretical values, indicating that probably all alpha 5 subunits coexist with gamma 2 subunits to form hippocampal GABAA receptors. Indeed, alpha 5gamma 2-containing receptors account for 21.8 ± 2.3% of hippocampal [3H]muscimol binding sites, a value similar to the 19 ± 4% of anti-gamma 2-immunoprecipitated [3H]L-655,708 binding sites and the [3H]L-655,708/[3H]Ro15-1788 ratio (15 ± 2%) (p > 0.40, one-way analysis of variance). Although the widely accepted stoichiometry of GABAA receptors is 2alpha 2beta 1gamma (for review, see McKernan and Whiting, 1996), quantitative immunoprecipitation and Western blot analysis have revealed the coexistence of gamma 2 and gamma 3 in approximately 7% of rat brain GABAA receptors (Quirk et al., 1994a). The pharmacological characteristics of these gamma 2gamma 3-containing isoforms have not been analyzed, and the possibility that the gamma 2 subunit is pharmacologically predominant over the gamma 3 subunit, thus masking the detection of alpha 5beta 3gamma 2gamma 3 complexes, cannot be excluded. Such a predominant effect has been shown for the alpha 1 subunit over the alpha 3 subunit in native cortical GABAA receptors (Araujo et al., 1996), but not over the alpha 6 subunit in cerebellar receptors (Khan et al., 1996).

The presence of low levels of [3H]L-655,708 binding associated with anti-alpha 1-, anti-alpha 2-, and anti-alpha 3-immunoprecipitated receptors suggested the existence of receptors with mixed contents of alpha  subunits. By combining the estimated amounts of alpha 1 (43%), alpha 2 (18%), and alpha 3 (17%) in rat hippocampus (McKernan and Whiting, 1996) and the [3H]L-655,708 data, it is possible to estimate that alpha 5alpha X=1,2,3 receptors account for ~15% of total alpha 5 subunit-containing receptors in rat hippocampus. Although these isoforms are of low abundance, it seems that the pharmacological characteristics of the alpha 5 subunit predominate, as judged by the binding of the alpha 5-selective ligand [3H]L-655,708. A contribution of each alpha  subunit to the overall receptor pharmacology has been shown in alpha 1alpha 6- and alpha 1alpha 3-containing receptors, with more or less predominance of one subunit over the other (Khan et al., 1996; Araujo et al., 1996).

In a recent study, Fritschy et al. (1997) showed that levels of alpha 5, beta 2/3, and gamma 2 subunit expression remain unaffected in gamma 3-deficient mutant mice, suggesting the coassembly of these proteins to form native GABAA receptors. The pharmacological and biochemical data reported here support and strengthen such a conclusion, because they demonstrate a preferential association of the alpha 5 subunit with the gamma 2 subunit in both rat and human hippocampus and establish alpha 5beta 2/3gamma 2 as a native hippocampal GABAA receptor isoform.

    Acknowledgments

We gratefully acknowledge the support of the Molecular Biology Department.

    Footnotes

Received May 14, 1998; Accepted June 26, 1998

Send reprint requests to: Dr. Cyrille Sur, Department of Biochemistry, Merck Sharp and Dohme Research Laboratories, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR, UK. E-mail: crrille_sur{at}merck.com

    Abbreviation

GABA, gamma -aminobutyric acid.

    References
Top
Summary
Introduction
Procedures
Results
Discussion
References

0026-895X/98/050928-06$3.00/0
MOLECULAR PHARMACOLOGY, 54:928-933 (1998).
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics


This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
S. S. Sarang, S. M. Lukyanova, D. D. Brown, B. S. Cummings, S. R. Gullans, and R. G. Schnellmann
Identification, Coassembly, and Activity of {gamma}-Aminobutyric Acid Receptor Subunits in Renal Proximal Tubular Cells
J. Pharmacol. Exp. Ther., January 1, 2008; 324(1): 376 - 382.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
E. Eugene, C. Depienne, S. Baulac, M. Baulac, J. M. Fritschy, E. Le Guern, R. Miles, and J. C. Poncer
GABAA Receptor {gamma}2 Subunit Mutations Linked to Human Epileptic Syndromes Differentially Affect Phasic and Tonic Inhibition
J. Neurosci., December 19, 2007; 27(51): 14108 - 14116.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
I. Marchionni, A. Omrani, and E. Cherubini
In the developing rat hippocampus a tonic GABAA-mediated conductance selectively enhances the glutamatergic drive of principal cells
J. Physiol., June 1, 2007; 581(2): 515 - 528.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
M. Mortensen and T. G. Smart
Extrasynaptic {alpha}{beta} subunit GABAA receptors on rat hippocampal pyramidal neurons
J. Physiol., December 15, 2006; 577(3): 841 - 856.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
S. K. Sullivan, R. E. Petroski, G. Verge, R. S. Gross, A. C. Foster, and D. E. Grigoriadis
Characterization of the Interaction of Indiplon, a Novel Pyrazolopyrimidine Sedative-Hypnotic, with the GABAA Receptor
J. Pharmacol. Exp. Ther., November 1, 2004; 311(2): 537 - 546.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. M. Petrini, I. Marchionni, P. Zacchi, W. Sieghart, and E. Cherubini
Clustering of Extrasynaptic GABAA Receptors Modulates Tonic Inhibition in Cultured Hippocampal Neurons
J. Biol. Chem., October 29, 2004; 279(44): 45833 - 45843.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
V. B. Caraiscos, E. M. Elliott, K. E. You-Ten, V. Y. Cheng, D. Belelli, J. G. Newell, M. F. Jackson, J. J. Lambert, T. W. Rosahl, K. A. Wafford, et al.
Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by {alpha}5 subunit-containing {gamma}-aminobutyric acid type A receptors
PNAS, March 9, 2004; 101(10): 3662 - 3667.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
N. Collinson, F. M. Kuenzi, W. Jarolimek, K. A. Maubach, R. Cothliff, C. Sur, A. Smith, F. M. Otu, O. Howell, J. R. Atack, et al.
Enhanced Learning and Memory and Altered GABAergic Synaptic Transmission in Mice Lacking the alpha 5 Subunit of the GABAA Receptor
J. Neurosci., July 1, 2002; 22(13): 5572 - 5580.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
H. Mohler, J. M. Fritschy, and U. Rudolph
A New Benzodiazepine Pharmacology
J. Pharmacol. Exp. Ther., January 1, 2002; 300(1): 2 - 8.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
C. Sur, K. A. Wafford, D. S. Reynolds, K. L. Hadingham, F. Bromidge, A. Macaulay, N. Collinson, G. O'Meara, O. Howell, R. Newman, et al.
Loss of the Major GABAA Receptor Subtype in the Brain Is Not Lethal in Mice
J. Neurosci., May 15, 2001; 21(10): 3409 - 3418.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
M. I. Strakhova, S. C. Harvey, C. M. Cook, J. M. Cook, and P. Skolnick