Characterization of the recombinant human neuronal nicotinic acetylcholine receptors alpha3beta2 and alpha4beta2 stably expressed in HEK293 cells.

HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations alpha3beta2 or alpha4beta2. [(3)H]-(+/-)Epibatidine ([(3)H]-(+/-)EPI) bound to membranes from A3B2 (alpha3beta2) and A4B2.2 (alpha4beta2) cells with K(d) values of 7.5 and 33.4 pM and B(max) values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca(2+) concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-beta-erythroidine (DHbetaE) in A3B2 cells and MEC=DHbetaE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in alpha3beta2 and alpha4beta2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca(2+), respectively. Our results indicate that stably expressed alpha3beta2 and alpha4beta2 human nAChRs are pharmacologically and functionally distinct.

2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist.

In the present paper we describe 2-methyl-6-(phenylethynyl)-pyridine (MPEP) as a potent, selective and systemically active antagonist for the metabotropic glutamate receptor subtype 5 (mGlu5). At the human mGlu5a receptor expressed in recombinant cells, MPEP completely inhibited quisqualate-stimulated phosphoinositide (PI) hydrolysis with an IC50 value of 36 nM while having no agonist or antagonist activities at cells expressing the human mGlu1b receptor at concentrations up to 30 microM. When tested at group II and III receptors, MPEP did not show agonist or antagonist activity at 100 microM on human mGlu2, -3, -4a, -7b, and -8a receptors nor at 10 microM on the human mGlu6 receptor. Electrophysiological recordings in Xenopus laevis oocytes demonstrated no significant effect at 100 microM on human NMDA (NMDA1A/2A), rat AMPA (Glu3-(flop)) and human kainate (Glu6-(IYQ)) receptor subtypes nor at 10 microM on the human NMDA1A/2B receptor. In rat neonatal brain slices, MPEP inhibited DHPG-stimulated PI hydrolysis with a potency and selectivity similar to that observed on human mGlu receptors. Furthermore, in extracellular recordings in the CA1 area of the hippocampus in anesthetized rats, the microiontophoretic application of DHPG induced neuronal firing that was blocked when MPEP was administered by iontophoretic or intravenous routes. Excitations induced by microiontophoretic application of AMPA were not affected.

SIB-1757 and SIB-1893: selective, noncompetitive antagonists of metabotropic glutamate receptor type 5.

Cell lines expressing the human metabotropic glutamate receptor subtype 5a (hmGluR5a) and hmGluR1b were used as targets in an automated high-throughput screening (HTS) system that measures changes in intracellular Ca2+ ([Ca2+]i) using fluorescence detection. This functional screen was used to identify the mGluR5-selective antagonist, SIB-1757 [6-methyl-2-(phenylazo)-3-pyridinol], which inhibited the glutamate-induced [Ca2+]i responses at hmGluR5 with an IC50 of 0.37 microM compared with an IC50 of >100 microM at hmGluR1. Schild analysis demonstrated a noncompetitive mechanism of inhibition. Pharmacophore mapping was used to identify an additional compound, SIB-1893 [(E)-2-methyl-6-(2-phenylethenyl)pyridine], which was also shown to block glutamate-induced increases in [Ca2+]i at hmGluR5 with an IC50 of 0.29 microM compared with an IC50 of >100 microM at hmGluR1. SIB-1757 and SIB-1893 showed little or no activity when tested for agonist and antagonist activity at the other recombinant human mGluR subtypes, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and N-methyl-D-aspartate receptors. In rat neonatal brain slices, SIB-1757 and SIB-1893 inhibited (S)-3,5-dihydroxyphenylglycine (DHPG)-evoked inositol phosphate accumulation in hippocampus and striatum by 60% to 80%, with a potency similar to that observed on recombinant mGluR5. However, in the cerebellum, a brain region with low mGluR5 expression, SIB-1757 failed to inhibit DHPG-evoked inositol phosphate accumulation. In cultured rat cortical neurons, SIB-1757 and SIB-1893 largely inhibited DHPG-evoked [Ca2+]i signals, revealing a population of neurons that were less sensitive to SIB-1757 and SIB-1893. This is the first description of highly selective, noncompetitive mGluR5 antagonists. These compounds will be useful tools in evaluating the role of mGluR5 in normal physiology and in animal models of disease.

The human N-methyl-D-aspartate receptor 2C subunit: genomic analysis, distribution in human brain, and functional expression.

cDNAs encoding four isoforms of the human NMDA receptor (NMDAR) NMDAR2C (hNR2C-1, -2, -3, and -4) have been isolated and characterized. The overall identity of the deduced amino acid sequences of human and rat NR2C-1 is 89.0%. The sequences of the rat and human carboxyl termini (Gly925-Val1,236) are encoded by different exons and are only 71.5% homologous. In situ hybridization in human brain revealed the expression of the NR2C mRNA in the pontine reticular formation and lack of expression in substantia nigra pars compacta in contrast to the distribution pattern observed previously in rodent brain. The pharmacological properties of hNR1A/2C were determined by measuring agonist-induced inward currents in Xenopus oocytes and compared with those of other human NMDAR subtypes. Glycine, glutamate, and NMDA each discriminated between hNR1A/2C-1 and at least one of hNR1A/2A, hNR1A/2B, or hNR1A/2D subtypes. Among the antagonists tested, CGS 19755 did not significantly discriminate between any of the four subtypes, whereas 5,7-dichlorokynurenic acid distinguished between hNR1A/2C and hNR1A/2D. Immunoblot analysis of membranes isolated from HEK293 cells transiently transfected with cDNAs encoding hNR1A and each of the four NR2C isoforms indicated the formation of heteromeric complexes between hNR1A and all four hNR2C isoforms. HEK293 cells expressing hNR1A/ 2C-3 or hNR1A/2C-4 did not display agonist responses. In contrast, we observed an agonist-induced elevation of intracellular free calcium and whole-cell currents in cells expressing hNR1A/2C-1 or hNR1A/2C-2. There were no detectable differences in the macroscopic biophysical properties of hNR1A/2C-1 or hNR1A/2C-2.

Pharmacological characterization of the human ionotropic glutamate receptor subtype GluR3 stably expressed in mammalian cells.

We have cloned the human ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR3 flip splice variant (hGluR3i) and developed a stable cell line expressing this receptor in HEK293 cells. Electrophysiological recordings demonstrated that glutamate-evoked currents desensitize rapidly, with a mean desensitization time constant of 5.4 ms. Robust glutamate-evoked increases in intracellular Ca++ ([Ca++]i) were observed in the presence of cyclothiazide, which attenuated receptor desensitization. [Ca++]i measurements were used to perform a detailed pharmacological characterization of hGluR3i with reference agonists and antagonists. The results of these studies showed that kainate and domoate were not fully efficacious agonists relative to glutamate. The binding affinities of agonists and competitive antagonists were determined in a [3H]AMPA competition binding assay. There was a good correlation between the functional data and the binding affinities obtained for competitive antagonists. However, the binding affinities of the agonists did not correlate with their functional EC50 values from [Ca++]i data, possibly because the binding assay predominantly measures the desensitized high-affinity state of the receptor. [3H]AMPA binding also was performed on membranes prepared from rat forebrain, and comparison of the data from HEK293 cells expressing hGluR3i and rat forebrain suggest that nearly all of the reference compounds show similar binding activities between the two membrane preparations, with the exception of fluoro-willardiine, kainate and 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2-3-dione (NBQX). These data suggest that cells stably expressing recombinant hGluR3i represent pharmacologically valid experimental systems to study human AMPA receptors.

The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents.

Neuronal nicotinic acetylcholine receptors (NAChRs) are pentameric ligand-gated ion channel receptors which exist as different functional subunit combinations which apparently subserve different physiological functions as indicated by molecular biological and pharmacological techniques. It is possible to design and synthesize novel compounds that have greater selective affinities and efficacies than nicotine for different NAChRs, which should translate into different behavioral profiles and therapeutic potentials. Examples of NAChR agonists studied are nicotine, SIB-1508Y, SIB-1553A and epibatidine. These compounds have different degrees of selectivity for human recombinant NAChRs, different neurotransmitter release profiles in vitro and in vivo and differential behavioral profiles. Preclinical studies suggest that SIB-1508Y is a candidate for the treatment of the motor and cognitive deficits of Parkinson's disease, whereas SIB-1553A appears to have potential as a candidate for the treatment of Alzheimer's disease. Epibatidine has a strong analgesic profile, however the ratio between pharmacological activity and undesirable effects is so low that it is difficult to envisage the use of this compound therapeutically. Nicotine has a broad profile of pharmacological activity, for instance demonstrating activity in models for cognition and analgesia. As for epibatidine, the adverse effects of nicotine severely limits its therapeutic use in humans. The discovery of subtype-selective NAChR agonists such as SIB-1508Y and SIB-1553A provides a new class of neuropsychopharmacological agents with better therapeutic ratios than nonspecific agents such as nicotine.

Functional characterization of human N-methyl-D-aspartate subtype 1A/2D receptors.

The human NMDAR2D subunit was cloned, and the pharmacological properties of receptors resulting from injection of transcripts encoding human NMDAR1A and NMDAR2D subunits in Xenopus oocytes were characterized by profiling NMDA receptor agonists and antagonists. We found that glutamate, NMDA, glycine, and D-serine were significantly more potent on hNMDAR1A/2D than on hNMDAR1A/2A or hNMDAR1A/2B. Also, the potencies of NMDA and glycine were higher for hNMDAR1A/2D than for hNMDAR1A/2C. Ifenprodil was more potent at hNMDAR1A/2B than at hNMDAR1A/2D, whereas 5,7-dichlorokynurenate was more potent at hNMDAR1A/2A than at hNMDAR1A/2D. As measured in transiently transfected human embryonic kidney 293 cells, the maximal inward current in the presence of external Mg2 occurred at -40 mV, and full block was not observed at negative potentials. Kinetic measurements revealed that the higher affinity of hNMDAR1A/2D for both glutamate and glycine relative to hNMDAR1A/2A and hNMDA1A/2B can be explained by slower dissociation of each agonist from hNMDAR1A/2D. The hNMDAR1A/2D combination represents a pharmacologically and functionally distinct receptor subtype and may constitute a potentially important target for therapeutic agents active in the human CNS.

Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells.

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.

Expression of N-methyl-D-aspartate receptor subunit mRNAs in the human brain: striatum and globus pallidus.

N-methyl-D-aspartate receptors (NRs) play an important role in basal ganglia function. By using in situ hybridization with ribonucleotide probes, we investigated the regional and cellular distribution of NR subunit mRNA expression in the human basal ganglia: caudate nucleus, putamen, lateral globus pallidus (LGP), and medial globus pallidus (MGP). Analysis of both film autoradiograms and emulsion-dipped slides revealed distinct distribution patterns for each subunit. On film autoradiograms, the signal for NR1, NR2B, and NR2C in the striatum (STR) was higher than in globus pallidus (GP). The NR2D probe gave a stronger signal in GP than in STR. For NR2A we found a signal in all regions. Analysis of emulsion-dipped sections demonstrated that in striatal neurons, the NR2B signal was higher than in GP neurons. In GP neurons, NR2D was more abundant than in striatal neurons. Despite the relatively low signal on film for NR2C in GP, we found a slightly higher signal in GP per neuron than in STR since in the pallidal areas neurons were sparse but intensely labeled. NR1 and NR2A were more evenly distributed over neurons of STR and GP Between the different parts of STR and GP, we observed only minor differences in the expression of NRs. In MGP a subpopulation of neurons exhibiting low NR2D signals could be separated from the majority of neurons showing an intense NR2D signal. Since the physiological properties of NRs are dependent on subunit composition, these data suggest a high degree of regional specialization of NR properties in the human basal ganglia.

Expression of N-methyl-D-aspartate receptor subunit mRNAs in the human brain: hippocampus and cortex.

N-methyl-D-aspartate receptor (NR) activation in the hippocampus and neocortex plays a central role in memory and cognitive function. We analyzed the cellular expression of the five NR subunit (NR1 and NR2A-D) mRNAs in these regions with in situ hybridization and human ribonucleotide probes. Film autoradiograms demonstrated a distinct pattern of hybridization signal in the hippocampal complex and the neocortex with probes for NR1, NR2A, and NR2B mRNA. NR2C and NR2D probes yielded scattered signals without a distinct organization. At the emulsion level, the NR1 probe produced high-density hybridization signals across the hippocampal complex. NR2A mRNA was higher in dentate granule cells and pyramidal cells in CA1 and subiculum compared to hilus neurons. NR2B mRNA expression was moderate throughout, with higher expression in dentate granule cells, CA1 and CA3 pyramidal cells than in hilus neurons. In the hippocampal complex, the NR2C probe signal was not different from background in any region, whereas the NR2D probe signal resulted in low to moderate grain densities. We analyzed NR subunit mRNA expression in the prefrontal, parietal, primary visual, and motor cortices. All areas displayed strong NR1 hybridization signals. NR2A and NR2B mRNAs were expressed in cortical areas and layers. NR2C mRNA was expressed at low levels in distinct layers that differed by region and the NR2D signal was equally moderate throughout all regions. Pyramidal cells in both hippocampus and neocortex express NR1, NR2A, NR2B, and, to a lesser extent, NR2D mRNA. Interneurons or granular layer neurons and some glial cells express NR2C mRNA.

Expression of N-methyl-D-aspartate receptor subunit mRNA in the human brain: mesencephalic dopaminergic neurons.

Evidence is accumulating that glutamate-mediated excitotoxicity plays an important role in neuronal degeneration in Parkinson's disease (PD). In addition, alterations in excitatory amino acid neurotransmission in the basal ganglia contribute to the clinical manifestations of motor dysfunction. However, detailed knowledge of the anatomical distribution and subtype specificity of glutamate receptors in the dopamine neurons of human substantia nigra (SN) has been lacking. In order to test the hypothesis that selective expression of particular N-methyl-D-aspartate receptor (NR) subunit mRNA contributes to the differential vulnerability of specific neuronal populations to excitotoxic injury in PD, we have used a quantitative dual label, in situ hybridization technique with ribonucleotide probes to examine the cellular distribution of NR subunit mRNA in postmortem human mesencephalic dopaminergic neurons from subjects with no known neurological disorder. Analysis of both film autoradiograms and emulsion-dipped sections demonstrated significant labeling of nigral neurons for each NR subunit. Neuronal labeling was most intense for the NR1 and NR2D subunits, with low level labeling for the remaining subunits. In addition, we examined four subregions of the ventral mesencephalon for differential expression of NR subunit mRNA. For all NR subunits, the pars lateralis (PL) exhibited the most intense signal, while neurons of the ventral tier substantia nigra pars compacta (SNpc) failed to demonstrate a preponderance of a particular subunit. These results demonstrate that NRs are expressed to a significant degree in dopaminergic neurons of the SN and that their distribution does not correlate with the characteristic pattern of neuronal degeneration in PD.

Cloning and stable expression of the mGluR1b subtype of human metabotropic receptors and pharmacological comparison with the mGluR5a subtype.

We isolated and characterized a cDNA encoding the human metabotropic glutamate receptor subtype 1b (hmGluR1b). In situ hybridization studies in human brain regions revealed a higher distribution of mGluR1 mRNA in the dentate gyrus of the hippocampus, the substantia nigra pars compacta and the Purkinje cell layer of the cerebellum compared to other regions studied. We established stable expression of recombinant hmGluR1b in L(tk-) mouse fibroblast and Chinese hamster ovary (CHO-dhfr-) cells. In both expression systems, agonist activation of hmGluR1b stimulated inositol phosphate (InsP) formation and elevation of the cytosolic free calcium ([Ca2+]i), and both responses were blocked by (S)-MCPG. The rank order of potency for agonists was quisqualate > glutamate > (1S,3R)-ACPD in both expression systems. Comparison of the agonist profiles of hmGluR1b and hmGluR5a, both stably expressed in L(tk-) cells, indicated the same rank order of potency (quisqualate > glutamate > or = (RS)-3,5-DHPG > or = (1S,3R)-ACPD), but each of the four agonists were more potent on hmGluR5a than on hmGluR1b. In antagonist studies, (S)-MCPG inhibited the agonist-induced InsP formation and elevation of [Ca2+]i in both hmGluR1b- and hmGluR5a-expressing cells. (S)-4CPG and (S)-4C3HPG both inhibited agonist responses only in hmGluR1b-expressing cells. However, in hmGluR5a-expressing cells the antagonist activity of (S)-4CPG and (S)-4C3HPG was dependent on the agonist used in the study, since they inhibited responses to glutamate but not to quisqualate. Stable cell lines expressing specific subtypes of human mGluRs represent valuable tools for the study of the mechanism of action of mGluRs at the molecular and cellular level and as screening targets for identification of subtype-selective agonists or antagonists.

Cellular distribution of NMDA glutamate receptor subunit mRNAs in the human cerebellum.

We have used a quantitative in situ hybridization method with human ribonucleotide probes to examine the regional and cellular distribution of N-methyl-D-aspartate receptor (NMDAR) subunit mRNAs in the human cerebellum. Purkinje cells showed very dense labeling for NMDAR1 mRNA, dense labeling for NMDAR2A mRNA, and moderate labeling for NMDAR2D mRNA, whereas labeling for NMDAR2C mRNA was low. Granule cells showed high hybridization signals for the NMDAR1 and NMDAR2C mRNAs and moderate signals for the NMDAR2A and NMDAR2D mRNAs. In addition intense labeling with the NMDAR2B probe was observed in medium-sized neurons with chromophilic cell bodies in the upper part of the granule cell layer, most likely representing Golgi cells. Neurons in the molecular layer, i.e., basket cells and stellate cells, showed moderate hybridization signals for NMDAR1 and NMDAR2D and low signal for NMDAR2C. Each type of cerebellar neuron analyzed displayed a distinct NMDAR2 subunit profile, suggesting that they are likely to have NMDA receptors with distinct properties.

Stable expression and characterization of recombinant human heteromeric N-methyl-D-aspartate receptor subtypes NMDAR1A/2A and NMDAR1A/2B in mammalian cells.

The electrophysiological and pharmacological properties of two mammalian cell lines stably transfected with cDNAs encoding recombinant human N-methyl-D-aspartate (NMDA) receptor subtypes NMDAR1A/2A and NMDAR1A/2B are described. In whole-cell electrophysiological recordings, application of NMDA/glycine elicited inward currents at negative holding potentials in human NMDAR1A/2A (hNMDAR1A/2A)- and hNMDAR1A/2B-expressing cells. The current-voltage relationships determined in both cell lines in the presence and absence of external Mg++ were similar to those observed with recombinant rat NMDA receptors. Power spectra calculated from NMDA/glycine-induced currents for both NMDA receptor-expressing cell lines suggested a kinetically homogeneous population of channels. Immunoprecipitation with an anti-NMDAR1A antibody coprecipitated the corresponding NMDAR2 subunit with the NMDAR1A, suggesting that heteromeric complexes are formed in these stable cell lines. Stimulation of NMDA receptors evoked an increase in intracellular Ca++, which was used to characterize their pharmacological properties. NMDA displayed less intrinsic activity than did glutamate in both NMDA receptor-expressing cell lines and was a 4-fold more potent agonist at hNMDAR1A/2B than hNMDAR1A/2A. NMDA/glycine-evoked increases in Ca++ levels were inhibited by CGS 19755, (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate, MK-801, ketamine and ifenprodil. (+/-)-3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonate was a 3-fold more potent antagonist at hNMDAR1A/2A than hNMDAR1A/2B, whereas ifenprodil was markedly more selective toward hNMDAR1A/2B, being 250-fold more potent than against hNMDAR1A/2A. These data suggest that cells stably expressing recombinant heteromeric hNMDAR1A/2A and hNMDAR1A/2B represent pharmacologically valid experimental systems to study human NMDA receptors.

Comparative structure of human neuronal alpha 2-alpha 7 and beta 2-beta 4 nicotinic acetylcholine receptor subunits and functional expression of the alpha 2, alpha 3, alpha 4, alpha 7, beta 2, and beta 4 subunits.

cDNA clones encoding human neuronal nicotinic acetylcholine receptor alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha 7, beta 2, beta 3, and beta 4 subunits were isolated from brainstem, hippocampus, prefrontal cortex, substantia nigra, thalamus, and IMR32 libraries. Human alpha 2 and alpha 6 and full-length beta 3 and beta 4 clones have not been previously reported. Deduced amino acid sequences of the alpha 2, alpha 6, beta 3, and beta 4 predicted mature peptides are 503 residues (56.9 kDa), 464 residues (53.7 kDa), 440 residues (50.8 kDa), and 477 residues (54.1 kDa), respectively. These sequences show 84 (alpha 2), 87 (alpha 6), 89 (beta 3), and 84% (beta 4) identity to the corresponding rat sequences. The amino termini of the human alpha 2 and beta 3 mature peptides contain 23 and six additional residues, respectively, compared to those of rat alpha 2 and beta 3. Recombinant receptors were expressed in Xenopus laevis oocytes injected with in vitro transcripts encoding either alpha 7 alone or alpha 2, alpha 3, or alpha 4 in pairwise combination with beta 2 or beta 4. Inward currents were elicited by the application of acetylcholine (1-100 microM) and other agonists; these responses were blocked 65-97% by application of 10 microM d-tubocurare, confirming functional expression of human nicotinic receptors.

Cloning and functional characterization of human heteromeric N-methyl-D-aspartate receptors.

Human cDNAs encoding N-methyl-D-aspartate receptor type (NMDAR)1A, NMDAR2A and NMDAR2B subunits were cloned and receptors encoded by these cDNAs were functionally expressed by injection of the respective mRNAs in Xenopus oocytes. The pharmacological properties of recombinant human N-methyl-D-aspartate (NMDA) receptors were characterized by profiling two agonists and four antagonists at both the NMDA and glycine sites in voltage-clamped oocytes. NMDA, glycine and D-serine were significantly more potent at human NMDAR (hNMDAR)1A/2B receptors than at nNMDAR1A/2A, whereas there was no detectable subtype-dependent difference in the potency of glutamate. Of the NMDA-site antagonists tested, CGP 43487 and 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonate exhibited 5.8- and 3.9-fold greater potency, respectively, at hNMDAR1A/2A receptors than at hNMDAR1A/2B. Of the four glycine-site competitive antagonists tested, L-689,560 displayed 5-fold greater potency at hNMDAR1A/2A, whereas 5,7-dichlorokynurenic acid, HA-966 and CGP 58411 did not discriminate between hNMDAR1A/2A and hNMDAR1A/2B. Receptors resulting from injection of hNMDAR1A, hNMDAR2A and hNMDAR2B transcripts in a 1:1:1 ratio were indistinguishable from hNMDAR1A/2B receptors in terms of their sensitivity to NMDA, glycine, D-serine, CGS 19755 and CGP 40116. Ifenprodil was approximately 350-fold more potent at hNMDAR1A/2B than at hNMDAR1A/2A receptors. Ifenprodil sensitivities of receptors formed in oocytes injected with a constant amount of hNMDAR1A mRNA but varying ratios of hNMDAR2A or hNMDAR2B mRNAs were compared. The receptors expressed at a 10:1 ratio of 2A:2B transcripts displayed an ifenprodil sensitivity that would be predicted for a population in which 51% was represented by hNMDAR(1A)2(2A)3 complexes. Our results underscore the need for subtype-selective compounds acting at novel sites to sufficiently probe the pharmacological differences between NMDA receptor subtypes formed by different subunit combinations.

Profiling of trans-azetidine-2,4-dicarboxylic acid at the human metabotropic glutamate receptors mGlu1b, -2, -4a and -5a.

We have tested the two enantiomers of trans-azetidine-2,4-dicarboxylic acid, (2S,4S)-azetidine-2,4-dicarboxylic acid ((2S,4S)-ADA) and (2R,4R)-azetidine-2,4-dicarboxylic acid ((2R,4R)-ADA) for activity at the human metabotropic glutamate receptors mGlu1b, mGlu2, mGlu4a and mGlu5a expressed in mammalian cells. In Chinese hamster ovary (CHO) cells expressing human mGlu2 receptors, 500 microM (2S,4S)-ADA inhibited forskolin-stimulated cAMP accumulation by 33 +/- 3% while 100 microM (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid induced an inhibition by 66 +/- 5%. The (2R,4R)-ADA enantiomer was inactive at human mGlu2 receptors. In CHO cells expressing human mGlu4a receptors, 10 microM L-AP4 inhibited forskolin-stimulated cAMP levels by 37 +/- 4% whereas both ADA enantiomers of trans-azetidine-2,4-dicarboxylic acid (500 microM) had no such effect. In CHO cells expressing human mGlu1b receptors and L cells expressing human mGlu5a receptors, both enantiomers, applied at 500 microM or 1 mM, were ineffective in stimulating inositolmonophosphate accumulation and did not affect quisqualate-stimulated inositolmonophosphate accumulation. We conclude that (2S,4S)-azetidine-2,4-dicarboxylic acid is a weak human mGlu2 receptor agonist and that (2R,4R)-azetidine-2,4-dicarboxylic acid is inactive at human mGlu2 receptors. Trans-azetidine-2,4-dicarboxylic acid has no significant agonistic effect on human mGlu4a receptors and neither agonistic nor antagonistic effects on human mGlu1b and mGlu5a receptors.

Human neuronal voltage-dependent calcium channels: studies on subunit structure and role in channel assembly.

Voltage-dependent calcium (Ca2+) channels, expressed in the CNS, appear to be multimeric complexes comprised of at least alpha 1, alpha 2 and beta subunits. Previously, we cloned and expressed human neuronal alpha 1, alpha 2 and beta subunits to study recombinant channel complexes that display properties of those expressed in vivo. The alpha 1B-mediated channel subtype binds omega-conotoxin (CgTx) GVIA with high affinity and exhibits properties of N-type voltage-dependent Ca2+ channels. Here we describe several alpha 2 and beta splice variants and report results on the expression of omega-CgTx GVIA binding sites, assembly of the subunit complex and biophysical function of alpha 1B-mediated channel complexes containing some of these splice variants. We optimized recombinant expression in human embryonic kidney (HEK) 293 cells of alpha 1B alpha 2b beta 1 subunit complexes by controlling the expression levels of subunit mRNAs and monitored cell surface expression by binding of omega-CgTx GVIA to the alpha 1B subunit. Co-expression of either alpha 2b or beta 1 subunits with an alpha 1B subunit increased expression of binding sites while the most efficient expression was achieved when both alpha 2b and beta 1 subunits were co-expressed with an alpha 1B subunit. The presence of alpha 2b affects the affinity of omega-CgTx GVIA binding and barium (Ba2+) current magnitudes, although it does not appear to alter kinetic properties of the Ba2+ current. This is the first evidence of an alpha 2 subunit modulating the binding affinity of a cell-surface Ca2+ channel ligand. Our results demonstrate that alpha 1, alpha 2 and beta subunits together contribute to the efficient assembly and functional expression of voltage-dependent Ca2+ channel complexes.