In 1989, a cDNA clone representing an EAA receptor channel was isolated fi*om a library of rat forebrain cDNA (Hollman et al., 1989). Upon transcription and expression
in Xenopus oocytes, the clone gave rise to a protein forming a functional receptor channel which possessed pharmacological and electrophysiological properties similar to the mammalian kainate receptor. Furthermore, the brain distribution of mRNA coding for this complex matched the distribution of kainate binding sites visualised by autoradiography (Gall et al, 1990; Keinanen et al, 1990). It was suggested that whilst the protein expressed in the oocytes may exist in vivo as a homo-oligomeric receptor- channel complex, it may also fimction as a subunit of hetero-oligomeric receptor-
channel complexes. In this way it would combine in vivo with other subunits to form native glutamate receptor-channel complexes with different characteristics (Hollman et al, 1989). Further probing has led to the identification of seven other mammalian non- NMDA subunits and two kainate binding proteins (KBPs)(Nakanishi and Masu, 1994). Based on amino acid sequence homology and pharmacological characteristics, the mammalian subunits have been grouped into three classes. The first group comprises the first four subunits discovered, GluRl to GluR4 (Hollman et al, 1989; Nakanishi et al, 1990; Keinanen et al, 1990; Boulter et al, 1990). Amino acids sequence homology in the four conserved membrane spanning domains of these receptors is between 80 and 90%. The other subunits, GluR5-GluR7 (Bettler et al, 1990; Bettler et al, 1992) show 30- 40% sequence homology with GluRl to GluR4 and so became members of a separate, second class of subunits (Egebjerg et al, 1991; Nakanishi and Masu, 1994). Kainate binding proteins KA-1 and KA-2 have been isolated from frog brain (Wada et al, 1989) and chick brain (Gregor et al, 1989), but did not form functional homomeric ion channels in transfected cells when expressed alone or in combination with subunits GluRl to GluR4 (Werner et al, 1991). GluR5 and GluR6 are homomeric channels activated by kainate but not AMPA. However, when either is co-expressed with KA-2, greater responses are recorded and the heteromeric channels respond to both AMPA and Kainate (Egebjerg and Heineman, 1993). In summary, it appears that the close structural homology of subunits GluRl to GluR4 parallels the similarities in their
functional characteristics; they give rise to receptor-channel complexes with properties more similar to those of AMPA receptors, (i.e. activation by AMPA followed by rapid desensitisation and competitive antagonism by CNQX at submicromolar concentration;
Keinanen et al, 1990). On the other hand, kainate is bound with much higher affinity by GluR6 which is incapable of binding AMPA. All these studies have shown that the pharmacological selectivity for kainate or AMPA and the resulting electrophysiological characteristics of the stimulated channels are dependent on the homomeric or heteromeric protein composition of the individual channels.
A functional metabotropic glutamate receptor has also been cloned, sequenced and expressed in Xenopus oocytes (Masu et al, 1991). Although similar in size to the ionotropic AMPA/kainate receptors, the overall sequence homology with these receptors is rather low. Similar amino acid sequences have been observed in both adrenoceptors and muscarinic receptors. More recently, 7 further subtypes of G-protein coupled metabotropic receptors have been described (mGluR 1 -mGlur8; Abe et al, 1992; Thomsen et al, 1992; Okamoto et al, 1994). Of these, mGluRl and mGluR5 are coupled to PI hydrolysis, mGluR2,3,4 and 6 have negative coupling to the cAMP cascade; however mGluR2 and mGluR3 interact with trans-ACPD, whereas mGluR4 and mGluR6 potently react with L-AP4 (Abe et al, 1992; Thomsen et al, 1992). Two new mGluR subtypes termed mGluR? and mGluRS have recently been characterized (Okamoto et al, 1994; Duvoisin et al. 1995).
Another recent development in the molecular biology of EAA receptors is the cloning of the NMDA receptor. In one report (Moriyoshi et al, 1991) a single protein which comprised 938 aminoacids residues was identified and cloned. Expression of this
protein, named NMDARl or NR-1, in Xenopus oocytes provided a homomeric receptor displaying rather low current amplitudes but with all the pharmacological properties characteristics of the native NMDA receptor, including blockade by
dizocilpine, Mg++, Zn++ and glycine antagonists. The amino terminus of the NMDARl (NR-1) sequence contains 56 glutamate and aspartate residues thus providing the high concentration of anionic sites necessary to facilitate Ca"^ permeabilty through the receptor channel. Another group (Kumar et al., 1991) reported the cloning in E. coli of one NMDA receptor subunit from rat brain, the glutamate-binding protein (GBP) which, like NR-1, has 4 membrane-spanning domains. Four further NMDA receptor proteins (NR-2A, NR-2B, NR-2C and NR-2D) have close similarity to each other but only 20% identity with NR-1 (Monyer et al., 1992; Hollmann and Heinemann, 1994). Co-expression of any one of the four with NR-1 gave 100 times greater responses in
Xenopus oocytes than channels composed of NR-1 only. In contrast to NR-1, none of the four other subunits (NR-2A to NR-2D) showed any receptor channel activity in a homomeric configuration or any heteromeric expression within the members of the NR- 2 subunits (Ishii et al., 1993) The mouse genome has generated three further NMDA receptor subunits (si, s2 and s3) which have 11-18% sequence identity with NR-1 (Meguro et al., 1992; Kutsuwada et al., 1992). When co-expressed with NR-1, each of these subunits give functional NMDA receptor channels activated by glycine, and different sensitivities to AP5, dizocilpine and 7-chlorokynurenic acid (Kutsuwada et al.,
1992). While s i has widespread distribution, s2 is found mainly in the forebrain and s3
in the cerebellum (Kutsuwada et al., 1992). These findings fit well with reports of NMDA receptors in different regions having slightly different pharmacology, binding characteristics and neurophysiology (Asher and Nowak, 1988b; Yoneda and Ogita
1.8. PHYSIOLOGICAL RELEVANCE OF EAA RECEPTOR