Functional assembly of AMPA and kainate receptors is mediated by several discrete protein-protein interactions.

Functional heterogeneity of ionotropic glutamate receptors arises not only from the existence of many subunits and isoforms, but also from combinatorial assembly creating channels with distinct properties. This heteromerization is subtype restricted and thought to be determined exclusively by the proximal extracellular N-terminal domain of the subunits. However, using functional assays for heteromer formation, we show that, besides the N-terminal domain, the membrane sector and the C-terminal part of S2 are critical determinants for the formation of functional channels. Our results are compatible with a model where the N-terminal domain only mediates the initial subunit associations into dimers, whereas for the assembly of the full functional tetramer, compatibility of the other regions is required.

A point mutation in the glutamate binding site blocks desensitization of AMPA receptors.

Desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors is thought to shape the synaptic response and act as a neuroprotective mechanism at central synapses, but the molecular mechanism underlying desensitization is poorly understood. We found that replacing the glutamate binding domain S1 of GluR3 (an AMPA receptor) with S1 of GluR6 (a kainate receptor) resulted in a fully active but completely nondesensitizing receptor. Smaller substitutions within S1 identified, besides two additional modulatory regions, a single exchange, L507Y, as is required and sufficient for the block of desensitization. This phenotype was specific for AMPA receptors and required an aromatic residue at this position. L507 lies between two residues (T504 and R509) that form part of the glutamate binding site. The physical proximity of these residues, which are involved in binding and gating, suggests they may form part of the link between these two events.

The tetrameric structure of a glutamate receptor channel.

The subunit stoichiometry of several ligand-gated ion channel receptors is still unknown. A counting method was developed to determine the number of subunits in one family of brain glutamate receptors. Successful application of this method in an HEK cell line provides evidence that ionotropic glutamate receptors share a tetrameric structure with the voltage-gated potassium channels. The average conductance of these channels depends on how many subunits are occupied by an agonist.

Sites of volatile anesthetic action on kainate (Glutamate receptor 6) receptors.

Molecular mechanisms of anesthetic action on neurotransmitter receptors are poorly understood. The major excitatory neurotransmitter in the central nervous system is glutamate, and recent studies found that volatile anesthetics inhibit the function of the alpha-amino-3-hydroxyisoxazolepropionic acid subtype of glutamate receptors (e.g. glutamate receptor 3 (GluR3)), but enhance kainate (GluR6) receptor function. We used this dissimilar pharmacology to identify sites of anesthetic action on the kainate GluR6 receptor by constructing chimeric GluR3/GluR6 receptors. Results with chimeric receptors implicated a transmembrane region (TM4) of GluR6 in the action of halothane. Site-directed mutagenesis subsequently showed that a specific amino acid, glycine 819 in TM4, is important for enhancement of receptor function by halothane (0. 2-2 mM). Mutations of Gly-819 also markedly decreased the response to isoflurane (0.2-2 mM), enflurane (0.2-2 mM), and 1-chloro-1,2, 2-trifluorocyclobutane (0.2-2 mM). The nonanesthetics 1, 2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane had no effect on the functions of either wild-type GluR6 or receptors mutated at Gly-819. Ethanol and pentobarbital inhibited the function of both wild-type and mutant receptors. These results suggest that a specific amino acid, Gly-819, is critical for the action of volatile anesthetics, but not of ethanol or pentobarbital, on the GluR6 receptor.

Agonist selectivity of glutamate receptors is specified by two domains structurally related to bacterial amino acid-binding proteins.

By exchanging portions of the AMPA receptor subunit GluR3 and the kainate receptor subunit GluR6, we have identified two discontinuous segments of approximately 150 amino acid residues each that control the agonist pharmacology of these glutamate receptors. The first segment (S1) is adjacent and N-terminal to the putative transmembrane domain 1 (TM1), whereas the second segment (S2) is located between the putative TM3 and TM4. Only the simultaneous exchange of S1 and S2 converts the pharmacological profile of the recipient to that of the donor subunit. The two segments identified in this study share sequence similarities with the ligand-binding site of several bacterial periplasmic amino acid-binding proteins. Based on the X-ray structure of these proteins, we propose a model for the glutamate-binding site of ionotropic glutamate receptors.

Differential assembly of coexpressed glutamate receptor subunits in neurons of rat cerebral cortex.

In the rat, subunits of the glutamate receptor family fall into three pharmacologically distinct groups: alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid preferring receptors (Glu R1-4), kainate preferring receptors (Glu R5-7, KA 1, KA 2), and N-methyl-D-aspartate preferring receptors (NMDA R1, NMDA R2A-2D). In the present study, we demonstrate immunocytochemically that the majority of neurons in rat cerebral cortex coexpress members of all three groups of glutamate receptor subunits, Glu R2/3, Glu R5/6/7, and NMDA R1. Using immunoaffinity purified or immunoprecipitated alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, kainate and N-methyl-D-aspartate receptors, we show that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors containing Glu R1-4, kainate receptors containing Glu R6, Glu R7, and KA 2 and N-methyl-D-aspartate receptors containing NMDA R1 each form distinct protein complexes that do not share subunits. Our data indicate that a mechanism exists which allows for the specific assembly of selected glutamate receptor subunits into functionally and structurally distinct heteromeric receptors.

Cloning and functional expression of a tetrabenazine sensitive vesicular monoamine transporter from bovine chromaffin granules.

Using oligonucleotide primers derived from the vesicular monoamine transporters sequences, a cDNA predicted to encode the bovine chromaffin granule amine transporter has been cloned (b-VMAT2). Surprisingly, its structure is more similar to the rat brain transporter (VMAT2), than to the rat adrenal counterpart (VMAT1). Unlike rat VMAT1, bovine VMAT2 appears to be expressed both in the adrenal medulla and the brain, as judged by Northern analysis. After modification/deletion of the seven amino acids at the N-terminus of the protein it was expressed in a functional form. The order of affinity of the bovine VMAT2 transporter to substrates is: serotonin > dopamine = norepinephrine > epinephrine. Also, the recombinant bovine adrenal transporter is highly sensitive to tetrabenazine, in sharp contrast to the rat adrenal transporter. The findings indicate, therefore, a clear species variation in which structure and function of the bovine adrenal transporter resemble the rat brain protein, while its tissue distribution is distinct from both types of rat proteins. In addition, the predicted protein sequence is identical to the experimentally determined N-terminus sequence of the purified vesicular amine transporter [Stern-Bach et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9730-9733].

Modification of arginyl or histidyl groups affects the energy coupling of the amine transporter.

We have characterized the effects of phenylglyoxal and diethyl pyrocarbonate (DEPC) on the catalytic cycle of the amine transporter in chromaffin granule membrane vesicles. Both reagents inhibited transport in a dose-dependent reaction (with IC50 values of 8 and 1 mM, respectively). The inhibition by DEPC was specific for histidyl groups since transport could be restored by treatment with hydroxylamine. Neither phenylglyoxal nor DEPC inhibited binding of either R1- or R2-type ligands, indicating that the inhibition of transport is not due to a direct interaction with either of the known binding sites. Interestingly, however, the acceleration of reserpine binding (an R1 ligand) by a transmembrane H+ gradient is inhibited by both reagents at concentrations identical to those which inhibit transprot. As previously demonstrated, transport of one proton across the transporter is required for this acceleration to take place [Rudnick, G., Steiner-Mordoch, S., Fishkes, H., Stern-Bach, Y., & Schuldiner, S. (1990) Biochemistry 29, 603-608]. Therefore, we suggest that either proton transport or a conformational change induced by proton transport is inhibited by both types of reagents.

Homology of a vesicular amine transporter to a gene conferring resistance to 1-methyl-4-phenylpyridinium.

The vesicular amine transporter (VAT) catalyzes transport and storage of catechol and indolamines into subcellular organelles in a wide variety of cells. It plays a central role in neurotransmission and is the primary target for several pharmacological agents. One of the drugs, reserpine, binds very tightly to the transporter and remains bound even after solubilization, a finding that has proven useful for purification of the transporter from bovine adrenal medulla in a fully functional state. The sequences of 26 N-terminal amino acids and of an additional 7-amino acid internal peptide are presented. Antibodies against a synthetic peptide based on the above sequences immunoprecipitate the transporter, confirming the conclusion that the peptide sequence is derived from bovine VAT. To our knowledge, documentation of sequences of vesicular neurotransmitter transporters has not been presented previously. In addition, the sequences obtained are highly homologous to the predicted sequence of a protein from PC12 cells that confers to Chinese hamster ovary cells resistance to 1-methyl-4-phenylpyridinium (MPP+), an agent that causes parkinsonism in model systems, confirming the hypothesis that the protein conferring resistance to MPP+ is a VAT.

Covalent modification of the amine transporter with N,N'-dicyclohexylcarbodiimide.

N,N'-Dicyclohexylcarbodiimide (DCC) has been previously shown to inhibit the amine transporter from chromaffin granules [Gasnier, B., Scherman, D., & Henry, J.P. (1985) Biochemistry 24, 3660-3667]. A study of the mechanism of inhibition is presented together with the demonstration of covalent modification of the protein. DCC inhibits binding of R1 (reserpine) and R2 (tetrabenazine) types of ligands to the transporter as well as transport. Ligands of the R2 type, but not those of the R1 type, protect against inhibition of all the reactions by DCC, i.e., accumulation of serotonin, binding if reserpine (R1 ligand), and binding of ketanserine (R2 ligand). The ability of a given R2 ligand to protect the transporter correlates well with its binding constant. Water-soluble carbodiimides, such as 1-ethyl-3-[3-(diethylamino)propyl]carbodiimide (EDC), do not have any effect on the catalytic activity of the transporter. A fluorescent hydrophobic analogue of DCC, N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4), inhibits at about the same concentration range as DCC. [14C]DCC labels several polypeptides in the chromaffin granule membranes. Labeling of a polypeptide with an apparent Mr of 80K is inhibited in the presence of R2 ligands. The labeled polypeptide copurifies with the recently identified and isolated transporter [Stern-Bach, Y., Greenberg-Ofrath, N., Flechner, I., & Schuldiner, S. (1990) J. Biol. Chem. 256, 3961-3966].

Identification and purification of a functional amine transporter from bovine chromaffin granules.

The amine transporter from bovine chromaffin granules has been purified in a functional state. Two isoforms with different pI values have been separated and shown to be active. One with an unusually acidic pI (approximately 3.5) has been shown to be a glycoprotein with an apparent Mr of 80,000. The purified polypeptide catalyzes transport of serotonin upon reconstitution with an apparent Km of 2 microM and a Vmax of 140 nmol/mg/min, 150-200-fold higher than the one determined in the native system. Transport is inhibited by reserpine and tetrabenazine, ligands which bind to two distinct sites on the transporter. These findings suggest that the binding sites for both drugs reside on a single polypeptide. The reconstituted purified transporter binds [3H]reserpine with a biphasic kinetic behavior, KD values of 0.3 and 30 nM and Bmax of 310 and 4200 pmol/mg protein, respectively. In addition, binding of [3H]reserpine is accelerated upon imposition of a pH gradient across the proteoliposome. From these findings it is evident that a single polypeptide catalyzes the various functions of the transporter.

Energetics of reserpine binding and occlusion by the chromaffin granule biogenic amine transporter.

The energetics of reserpine binding to the bovine adrenal biogenic amine transporter suggest that H+ ion translocation converts the transporter to a form which binds reserpine essentially irreversibly. Reserpine binding to bovine adrenal chromaffin granule membrane vesicles is accelerated by generation of a transmembrane pH difference (delta pH) (interior acid) or electrical potential (delta psi) (interior positive). Both components of the electrochemical H+ potential (delta mu H+) must be dissipated to block reserpine binding, and generation of either one stimulates the binding rate. Reserpine binding is less dependent than amine transport on the delta pH, suggesting that translocation of fewer H+ ions is required to expose the high-affinity site than are required for net transport. Bound reserpine dissociates very slowly, if at all, from the transporter. Binding is stable to 1% cholate, 1.5% Triton X-100, 1 M SCN-, and 8 M urea, but sodium dodecyl sulfate (0.035%) and high temperatures (100 degrees C) released bound reserpine, indicating that binding is noncovalent. The results raise the possibility that the transporter, by translocating one H+ ion outward down its concentration gradient, is converted to a form that can either transport a neutral substrate molecule inward or occlude reserpine in a dead-end complex.