Plasticity of agonist binding sites in hetero-oligomers of the unitary glutamate receptor subunit XenU1.

Two subunits from Xenopus, XenNR1G and the "short" subunit XenU1, have previously been coexpressed to form a unitary (NMDA/non-NMDA type) glutamate receptor. We now show that an antibody to XenNR1G or an antibody to XenU1 precipitates the binding sites of both XenNR1G and XenU1, with the recombinant subunits or with solubilised Xenopus brain membranes, i.e., the combination occurs in vivo. The expressed XenU1 subunits are in the cell membrane and oriented correctly. XenU1 binds not only kainate with high affinity (K(D) 1.2 nM at 25 degrees C), but also the glycine site antagonist 5,7-dichlorokynurenic acid (DCKA). DCKA, GTP, or GTPgammaS displaces competitively all of the bound [3H]kainate, but glycine has no effect. The results suggest that a common binding site for kainate, DCKA, and GTP can exist on XenU1. In the XenNR1G/XenU1 complex, the kainate affinity is lowered eightfold, whereas the DCKA affinity is considerably increased (K(D) 147 nM). Only 18% of the binding to the complex has the properties of the NMDA receptor glycine site, the rest being due to switching of the high-affinity kainate site of XenU1 (low-affinity DCKA) to a high-affinity DCKA (low-affinity kainate) conformation. Surprisingly, a mammalian NR2 subunit can also combine with XenU1, and this introduces similar reciprocal changes in the binding of kainate and DCKA. The combined evidence suggests a common basic mode of agonist site formation in different subunit types of the ionotropic glutamate receptors.

A unitary non-NMDA receptor short subunit from Xenopus: DNA cloning and expression.

A high-affinity homomeric, non-NMDA glutamate receptor was previously purified from the amphibian Xenopus laevis. We have obtained nine peptide sequences from its subunit, applied in cDNA cloning. The cDNA encodes a subunit (XenU1) containing all nine sequences. The 51,600-dalton mature subunit has four hydrophobic domains homologous to the four in the C-terminal half of mammalian non-NMDA receptor subunits. Transient expression in COS cells showed 1:1 binding (at Bmax) of [3H] kainate (KD = 9.1 nM) and of [3H] AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid; KD = 62 nM). The competitive binding series domoate > kainate > AMPA > NBQX > glutamate was established (where NBQX is 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo (f) quinoxaline). Each agonist shows the same KI value against [3H] kainate and [3H] AMPA binding, suggesting a common agonist site, but two conformations thereof are distinguishable by their different affinities for the antagonist NBQX and by the allosteric effect of thiocyanate anion (greatly potentiating AMPA binding, inert with kainate). XenU1 is exceptional among non-NMDA receptor subunits because it lacks most of the large N-terminal domain found in those of mammals and it has high affinity for both kainate and AMPA. It differs from the similarly-short "kainate-binding proteins" (KBPs), in binding AMPA and in forming glutamate receptor channels when the native protein is reconstituted. Moreover, whereas a full-length kainate receptor of mammals, GluR6, is shown here (from a partial cDNA sequence) to exist also in Xenopus, with approximately 97% sequence identity to rat GluR6, XenU1 is much less homologous to any rat kainate or AMPA receptor and also to the KBPs, even from another amphibian, Rana. Another difference is that a potential concensus sequence ("EF hand") for Ca2+ binding is present in the N-terminal domain of XenU1, but not in the chicken (glial) KBP. XenU1 is deduced to be in a new family of non-NMDA receptors.

Reconstitution of glutamate receptor proteins purified from Xenopus central nervous system into artificial bilayers.

Excitatory amino acid (EAA) receptor (EAAR) proteins purified from Xenopus central nervous system using a domoate affinity column and then separated into fractions using sucrose density gradient centrifugation were reconstituted, first into liposomes and then into planar lipid bilayers, using pipette-dipping and black lipid membrane techniques. Although the protein was eluted from the column with either alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) or kainate and could not be eluted with N-methyl-D-aspartate (NMDA), channel openings were obtained after exposure of the bilayers to kainate, AMPA, or NMDA (usually only in the presence of glycine). In bilayers exhibiting a single open channel conductance level this was approximately 6 pS with AMPA, approximately 9 pS with kainate, and approximately 50 pS with NMDA. However, with a few batches of protein unitary channel openings of up to 400 pS were observed, suggesting that reconstituted EAAR may sometimes form functional aggregates. The protein eluted from the domoate column was divided into two fractions on a sucrose density gradient. After reconstitution, one fraction responded to all three EAAs, whereas the other responded only to the non-NMDA receptor agonists. An explanation for these results is that some of the EAAR eluted from the column contain NMDA receptor subunits in addition to non-NMDA receptor subunits.

Purified unitary kainate/alpha-amino-3-hydroxy-5-methylisooxazole-propionate (AMPA) and kainate/AMPA/N-methyl-D-aspartate receptors with interchangeable subunits.

We have purified and characterized two vertebrate excitatory amino acid ionotropic receptors from the Xenopus central nervous system. Each is a unitary receptor (i.e., having more than one class of excitatory amino acid agonist specificity within one protein oligomer). The first is a unitary non-N-methyl-D-aspartate (non-NMDA) receptor and the second is a unitary NMDA/non-NMDA receptor. The specific agonist-activated channel activity and pharmacology of each type were recognized by patch-clamping lipid bilayers in which the isolated protein was reconstituted. In the second case, the NMDA and the non-NMDA sites could not be physically separated and exhibited functional interaction. Parallel evidence for this was obtained when poly(A) RNA from Xenopus brain was translated in oocytes: a noncompetitive inhibition of the response to L-kainate is produced by NMDA to a maximum depression of 30% at 1 mM NMDA. Each isolated oligomer contains 42-kDa subunits of the non-NMDA ligand binding type, but the second type has an additional NMDA-receptor-specific 100-kDa subunit. Thus, a subunit-exchange hypothesis can account for the known multiplicity of excitatory amino acid receptor types.

Kinetics and physical parameters of rat brain opioid receptors solubilized by digitonin and CHAPS.

Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The yield of solubilization was 70-75% with digitonin and 30-35% with CHAPS. Kinetic and equilibrium studies performed from digitonin extracts resulted in KD values comparable with those of the membrane fractions. Two [3H]naloxone binding sites were obtained in the extracts similarly to membrane fractions. The rank order potency of drugs used in the competition experiments did not change during solubilization. The distributions of mu, delta, and kappa opioid receptor binding sites were similar in membrane and digitonin-solubilized fractions (48-50% mu, 35-37% kappa, and 13-17% delta subtypes). The hydrodynamic properties of digitonin- and CHAPS-solubilized preparations were studied by sucrose density gradient centrifugation and Sepharose-6B chromatography. In all cases, two receptor populations were identified with the following parameters: sedimentation coefficients for the digitonin extracts were 9.2S and 13.2S and for CHAPS extract 8S and 15.6S; the Stokes radii were 45 A and 65A for the digitonin extract and 31A and 76A for the CHAPS-solubilized preparation.