c. Subunits Within the Pentamer
II. A Constrained Combinatorial System for the Receptor Compositions
We therefore start from the situation that a repertoire of at least 20 mam-malian subunits (including the y2 splice variant) is available. The total is 21 if the splice variant of
f32,
found to be expressed in the chicken (HARVEY et al.1994), is included, its origin in the TM3/TM4 loop being similar to the splic-ing there of y2, which is known (see above) to occur both in birds and mammals; indeed, two polypeptide forms of
f32
have also been found (although not yet sequenced) in the mammal (BENKE et al. 1994). This set is drawn upon to a total of five for each receptor molecule. The selection for this produces a combinatorial system for constructing these receptors. This could in principle generate an impossibly large number of subtypes: constraints which reduce this exist at several levels. The first is the separation into two pools for co-assembly as described above. This removes from the potential total the com-binations of the p subunits with any of the others. The second is that, so far as is known, all normal GABAA receptor molecules other than Aor forms (i) require both a and f3 subunits; (ii) require in addition one or more of the y, 8,E, nor 8 subunit types, which do not occur otherwise; (iii) usually contain either three or four different subunit types (which may include dual isoforms of one type, e.g. a1a6). A possible exception to (ii) might be receptors containing only a and f3 subunits: in vitro these can robustly co-express functional receptors in all host cell types tried (SCHOFIELD et al. 1987; SHIVERS et al. 1989; PUlA et al. 1991; SHINGAI et al. 1991; ANGELOTTI and MACDONALD 1993; ATKINSON
et al. 1994; HARTNETI et al. 1996), which behave as pentamers (KNIGHT et al.
1999) and which can be maintained long-term in stable cell lines (Moss et al.
1990; HADINGHAM et al. 1992). It is unknown, however, whether such
af3
recep-tors exist in vivo, though there is some evidence that they might in the case of an a413
receptor (BENCSITS et al. 1999).Binary combinations other than
af3
pairs, or even single subunits, can, for most of the a,13
or risoforms, also be expressed functionally, in oocytes and in some but not all (ANGELOTII et al. 1993) transfected mammalian cell types (BLAIR et al. 1988; PRITCHETI et al. 1988; SHIVERS et al. 1989; SIGEL et al. 1990;VERDOORN et al. 1990; SANNA et al. 1995; KRISHEK et al. 1996). This expression is in most cases weak, depending on the subtype, species or host cell, and it is always much increased when supplemented to give an
af3r
combination. It is not considered, therefore, to give an exception to the aforementioned rules operating in vivo. The selection foraf3ris
such that even the robustly expressedaf3
form disappears when a r subunit is added (ANGELOTII and MACDONALD 1993).Considering rule (ii), in the great majority of brain receptors it is a r subunit that complements a and
13,
as deduced from immunocytochemical and co-immunoprecipitation evidence (for references see BARNARD et al. 1998).That evidence also shows that r2 is by far the most abundant and ubiquitous of the GABAA receptor subunits in the CNS; by immunogold labelling the }2 subunit is very commonly seen localised at the same synaptic junction as a and
13
subunits (SOMOGYI et al. 1996; NUSSER et al. 1998). The dominance of af3rtypes is also shown by the high percentage of the native GABAA recep-tors sensitive to benzodiazepine (BZ) drugs, for which an af3rcombination is required. The far-reaching effects on the receptor population of the deletion of r subunits in transgenic mice are described by H. MOHLER (Chap. 3, this volume).In the limit of rule (iii), the theoretical maximum of different subunit types or isoforms combined in one molecule is five; analysis of extracted cerebellar GABAA receptors using several isoform-specific antibodies in turn (JECH-LINGER et al. 1998) gave results that were compatible with this maximum of five types occurring in certain very limited cases. These rules are derived from a large body of observations on the formation of functional receptors in het-erologous expression or on analyses of co-occurrence of subunits in receptors in or from native tissues.
That set of requirements arises, of course, from the types of interaction which can occur between the surfaces of different subunits. The interactions of the subunits which are thus selected must be energetically favourable for the assembly, the correct targeting and the stability of the active receptor.
Mostly the structural barriers to that correct assembly must be low, since any ternary combination of the
af3r
(i.e. lXj+!3j+n) form tested so far can interact in some or other host cell to produce a functional receptor, apparently self-directed to a single type (examples in SIGEL et al.1990; HADINGHAM et al.1992;ANGELOTII and MACDONALD 1993; SAXENA and MACDONALD 1994; DUCIC et al.
The Molecular Architecture of GABAA Receptors 87 1995; KIRSCH et al. 1995; SIEGHART 1995; WAFFORD et al. 1996; NEELANDS et al.
1999). This denotes high complementarity of the tertiary structures of three diverse subunit types, since (as reviewed above) homomers are strongly dis-favoured. The exception is the p class of subunits, and these differ from the others (as tested in a and
13)
in a determinant in the N-terminal domain (HACKAM et al.1998) which directs the interactions for separate assembly from the aforementioned two pools.If the only constraint on assembly when a,
13
and y subunits are present is that all those three types must co-assemble, then a possible total of 96 af3r-containing mammalian receptors could be created in this sub-class.The evidence on the native BZ-sensitive receptors suggests that their multi-plicity, although considerable, is well below this. Obviously a further constraint is the local gene expression program, since some theoretical partners will not co-occur in the same cells. For example, a6, a4, 8 or y3 have not been found with certain others. A second level of constraint here is that of the targeting or chaperone or anchoring mechanisms, which can direct subunit selection in the targeting or localisation or synaptic clustering (e.g. via gephyrin) of GABAA receptors (CRAIG et al. 1996; ESSRICH et al. 1998;
KNEUSSEL et al. 1999). Intermediate complexes which are not permissive for a preferred path of receptor assembly become degraded. That topic cannot be reviewed here, but it should be noted that for the GABAA receptors such processing in heterologous expression may not be a guide to its path in the neurones and may also differ between neuronal types, determined by the avail-abilities of specific controlling factors (as just noted). In vitro it has been found to vary for some GABAA receptor subunits even between different host cells. An example of more selective pairing in situ than in recom-binant expression is given by the set of aI, a6 and 8 subunits. The recombi-nant a1 and 8 subunits assemble well with
13
subunits to form functional receptors in each of three host systems used (SAXENA and MACDONALD 1994;DUCIC et al. 1995; KRISHEK et al. 1996). However, although those three subunit types co-exist in the same cerebellar granule cell, 8 is replaced by y2 in the receptors there which contain a1 (alone) plus a
f3
subunit, 8 always being combined instead with a6. This was shown by a variety of appro-aches: comprehensive immunogold localisations (NUSSER et al. 1998), co-immunopurifications (QUIRK et al. 1994b; JECHLINGER et al. 1998), an immunol freeze-fracture technique (CARUNCHO and COSTA 1994) and again by a6 trun-cation through gene targeting, which is found to deplete cerebellar a6 and 8 subunits together (JONES et al. 1997). This illustrates the additional level of constraint on the receptor compositions that can be exerted by processing in situ.The a,