Metal Ion and Guanine Nucleotide Modulations of Agonist Interaction in G-Protein-Coupled Serotonin1A Receptors from Bovine Hippocampus

1. The serotonin type 1A (5-HT_1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to GTP-binding regulatory proteins (G-proteins). We have studied the modulation of agonist binding to 5-HT_1A receptors from bovine hippocampus by metal ions and guanine nucleotide.2. Bovine hippocampal membranes containing the 5-HT_1A receptor were isolated. These membranes exhibited high-affinity binding sites for the specific agonist [³H]OH-DPAT.3. The agonist binding is inhibited by monovalent cations Na⁺, K⁺, and Li⁺ in a concentration-dependent manner. Divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺, on the other hand, show more complex behavior and induce enhancement of agonist binding up to a certain concentration. The effect of the metal ions on agonist binding is strongly modulated in the presence of GTP--S, a nonhydrolyzable analogue of GTP, indicating that these receptors are coupled to G-proteins.4. To gain further insight into the mechanisms of agonist binding to bovine hippocampal 5-HT_1A receptors under these conditions, the binding affinities and binding sites have been analyzed by Scatchard analysis of saturation binding data. Our results are relevant to ongoing analyses of the overall regulation of receptor activity for G-protein-coupled seven transmembrane domain receptors.     

Binding thermodynamics of serotonin to rat-brain 5-HT1a, 5HT2a and 5-HT3 receptors

The thermodynamic parameters ΔG° , ΔH° and Δs° of the binding equilibrium of serotonin to 5-HT_1A, 5-HT_2A and 5-HT₃ rat-brain membrane receptors have been determined by means of affinity constant measurements at six temperatures in the range 0 –35 ° C and van't Hoff plots. At variance with 5-HT_1A and 5-HT₃, the binding at the 5-HT_2A receptors is strongly endothermic and entropy-driven. Comparison with the results obtained by other authors on 5-HT_2A receptors in rats and humans suggests that the observed differences can be explained by a single amino acid difference in the receptor sequence between these two species.     

Role of Disulfides and Sulfhydryl Groups in Agonist and Antagonist Binding in Serotonin1A Receptors from Bovine Hippocampus

SUMMARY 1. The serotonin_1A (5-HT_1A) receptors are members of a superfamily of seven-transmembrane-domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. Mutagenesis and modeling studies point out that the ligand-binding sites in serotonin receptors are located in the transmembrane domain. However, these binding sites are not very well characterized. Since disulfide bonds and sulfhydryl groups have been shown to play vital roles in the assembly, organization, and function of various G-protein-coupled receptors, we report here the effect of disulfide and sulfhydryl group modifications on the agonist and antagonist binding activity of 5-HT_1A receptors from bovine hippocampus.2. DTT or NEM treatment caused a concentration-dependent reduction in specific binding of the agonist and antagonist in 5-HT_1A receptors from bovine hippocampal native and solubilized membranes. This is supported by a concomitant reduction in binding affinity.3. Pretreatment of the receptor with unlabeled ligands prior to chemical modifications indicate that the majority of disulfides or sulfhydryl groups that undergo modification giving rise to inhibition in binding activity could be at the vicinity of the ligand-binding sites.4. In addition, ligand-binding studies in presence of GTP--S, a nonhydrolyzable analogue of GTP, indicate that sulfhydryl groups (and disulfide bonds to a lesser extent) are vital for efficient coupling between the 5-HT_1A receptor and the G-protein.5. Our results point out that disulfide bonds and sulfhydryl groups could play an important role in ligand binding in 5-HT_1A receptors.     

Photoaffinity Labelling and Solubilization of the Central 5-HT1A Receptor Binding Site

Abstract

Two complementary approaches, covalent labelling and solubilization, have been used to study the biochemical properties of the central 5-HT_1A receptor binding site. We have first designed a photoaffinity ligand containing the structure of 8-OH-DPAT, a potent and specific agonist of 5-HT_1A sites. Thus, 8-methoxy-2[N-n-propyl,N-3-(2-nitro-4-azido-phenyl)- aminopropyl]aminotetralin or 8-methoxy-3'-NAP-amino-PAT, was found to displace, in the dark, [³H]8-OH-DPAT from 5-HT_1A sites in rat hippocampal membranes with an IC₅₀ of 6.6 nM. Under two cumulative UV irradiations (366 nm, for 20 min at 4°C), 8-methoxy-3-'-NAP-amino-PAT (30 nM) blocked irreversibly 55-60% of 5-HT_1A binding sites. This blockade was specific of 5-HT_1A sites since the other serotoninergic sites, 5-HT_1B, 5-HT₂ and also the presynaptic 5-HT₃ sites were not affected by the treatment. In addition, the binding of [³H]Spiperone and [³H]7-OH-DPAT to striatal dopamine sites remained unchanged under similar photolysis conditions. The tritiated derivative of the photoaffinity ligand (92 Ci/mmol) was then synthesized for the identification of the covalently bound protein(s). SDS-PAGE of solubilized membranes irradiated in the presence of 20 nM ³H-8-methoxy-3'-NAP-amino-PAT allowed the detection of a 63 kD protein whose labelling appeared specific. Thus, ³H-incorporation into the 63 kD band could be prevented by uM concentrations of 5-HT, 8-OH-DPAT and other selective 5-HT_1A ligands such as isapirone. In contrast, the 5-HT₂ antagonist ketanserin, norepinephrine and dopamine-related ligands (including 7-OH-DPAT) were ineffective. Direct solubilization of 5-HT_1A receptor binding sites was also attempted from rat hippocampal membranes. The best results were obtained using CHAPS (10 mM) plus NaCl (0.2 M), which led to 50 % recovery of 5-HT_1A sites in the 100,000 g supernatant. The pharmacological properties and sensitivity to N-ethyl-maleimide and GppNHp of soluble sites appeared near identical to those of membrane-bound 5-HT_1A sites.     

BMY 7378: Partial agonist at spinal cord 5-HT1A receptors

Recent data indicate that BMY 7378 demonstrates high affinity, selectivity and low intrinsic activity at hippocampal 5-HT_1A receptors, suggesting that BMY 7378 may represent the first selective 5-HT_1A functional antagonist. The present study examined the agonist and antagonist properties of BMY 7378 at spinal cord 5-HT_1A receptors. In electrophysiological studies, iontophoretic administration of either the 5-HT_1A agonist 8-OH-DPAT (43.8 ± 5.4 nA) or BMY 7378 (46.3 ± 5.2 nA) significantly inhibited the firing rate of wide-dynamic-range dorsal horn units indicating that BMY 7378 demonstrates significant intrinsic activity at spinal cord 5-HT_1A receptors. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 ejection current (20–100 nA) which antagonized the 8-OH-DPAT induced inhibition of dorsal horn unit activity. In behavioral studies in the spinal rat, 8-OH-DPAT increased the animals' sensitivity to noxious levels of mechanical stimulation (ED₅₀ = 269 ± 24 nmol/kg) as did BMY 7378 (ED₅₀ = 295 ± 70 nmol/kg) with no statistically significant difference in the maximal response (Y_max) observed. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 dose (10–1100 nmol/kg) which blocked the hyperalgesic effect of 8-OH-DPAT, rather, each drug produced similar effects which were additive. Further, the 5-HT_1A agonist effects of BMY 7378 were blocked by the 5-HT_1A antagonist, spiperone. Therefore, both the electrophysiologic and reflex data indicate that BMY 7378 possesses significant intrinsic activity at spinal cord 5-HT_1A receptors, and like 8-OH-DPAT is a partial agonist at these receptors. Differences in BMY 7378 intrinsic activity at spinal cord as opposed to hippocampal 5-HT_1A receptors are discussed in terms of regional differences in G-proteins coupled to 5-HT_1A receptors in these two CNS regions.     

Temperature-dependent interaction of the bovine hippocampal serotonin(1A) receptor with G-proteins

The ligand binding and G-protein coupling of the bovine hippocampal 5-HT1A receptor as a function of temperature was monitored. There is an almost complete and irreversible loss in agonist binding at 50 degrees C. However, the antagonist binding is reduced only by 50%, and this could be reversed if the temperature is lowered to 25 degrees C. Interestingly, the agonist binding of the 5-HT1A receptor in membranes exposed to 50 degrees C is inhibited to a much lesser extent by GTP-gamma-S, a non-hydrolysable analogue of GTP, indicating uncoupling of the 5-HT1A receptor to G-proteins at 50 degrees C. We propose that high temperature selectively and irreversibly inactivates G-proteins thereby affecting G-protein-receptor interaction and agonist binding of the 5-HT1A receptor.     

The cholesterol-complexing agent digitonin modulates ligand binding of the bovine hippocampal serotonin1A receptor

The serotonin_1A (5-HT_1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding of the bovine hippocampal 5-HT_1A receptor by cholesterol complexation in native membranes using digitonin. Complexation of cholesterol from bovine hippocampal membranes using digitonin results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT and antagonist p-MPPF to 5-HT_1A receptors. The corresponding changes in membrane order were monitored by analysis of fluorescence polarization data of the membrane depth-specific probes, DPH and TMA-DPH. Taken together, our results point out the important role of membrane cholesterol in maintaining the function of the 5-HT_1A receptor. An important aspect of these results is that non-availability of free cholesterol in the membrane due to complexation with digitonin rather than physical depletion is sufficient to significantly reduce the 5-HT_1A receptor function. These results provide a comprehensive understanding of the effects of the sterol-complexing agent digitonin in particular, and the role of membrane cholesterol in general, on the 5-HT_1A receptor function.     

Bell-shaped agonist activation of 5-HT1A receptor-coupled Gαi3 G-proteins: Receptor density-dependent switch in receptor signaling

A previous study observed bell-shaped concentration-response isotherms for activation of Gα_i3 G-protein subunits by high efficacy 5-HT_1A receptor agonists in a Chinese hamster ovary (CHO) cell line expressing high levels of these receptors. This suggested that a signaling switch took place in that cell line (from Gα_i3 to activation of other G-proteins) but it was unclear if such effects are observed for 5-HT_1A receptors in other cellular environments.Here, using an antibody capture-based [³⁵S]GTPγS binding assay for Gα_i3 activation, we investigated whether efficacious 5-HT_1A receptor agonists (5-HT, F13714, befiradol, NLX-101), prototypical agonists ((+) and (−)8-OH-DPAT), and partial agonist, antagonists, inverse agonists (pindolol, WAY100635, spiperone) produced similar effects on 5 cell lines expressing different levels of human 5-HT_1A receptors.In membranes from cell lines (HeLa, C6-glia and CHO-low) expressing moderate receptor levels (between 1 and 4 pmol/mg of protein), 5-HT, F13714, befiradol and NLX-101 elicited classical sigmoid concentration-response isotherms. In contrast, in cell lines (CHO-high, HEK-293F) expressing high receptor levels (>9 pmol/mg) these agonists elicited bell-shaped concentration-response isotherms that peaked at nanomolar-range concentrations and then returned to baseline or below. Spiperone elicited inverse agonist inhibitory sigmoid isotherms in all membrane preparations while WAY100635 was mostly ‘silent’ for Gα_i3 activation. The other compounds elicited diverse responses in the different cell lines suggesting that other factors, in addition to receptor expression levels, could be influencing Gα_i3 activation.These data indicate that Gα_i3 G-protein activation by 5-HT_1A receptor ligands is highly dependent on receptor expression levels and on cellular background. Moreover, the induction of bell-shape concentration-response isotherms by 5-HT and other high-efficacy agonists is consistent with a switch in signaling to other G-protein-mediated signaling cascades, possibly elicited by receptor conformational changes.     

Synthesis of novel 5-substituted-2-aminotetralin analogs: 5-HT1A and 5-HT7 G protein-coupled receptor affinity, 3D-QSAR and molecular modeling

The serotonin 5-HT₇ G protein-coupled receptor (GPCR) is a proposed pharmacotherapeutic target for a variety of central and peripheral indications, albeit, there are no approved drugs selective for binding 5-HT₇. We previously reported that a lead analog based on the 5-substituted-N,N-disubstituted-1,2,3,4-tetrahydronaphthalen-2-amine (5-substituted-2-aminotetralin, 5-SAT) scaffold binds with high affinity at the 5-HT₇ GPCR, and can treat symptoms of autism in mouse models; subsequently, the lead was found to have high affinity at the 5-HT_1A GPCR. Herein, we report the synthesis of novel 5-SAT analogs to develop a 3-dimensional quantitative structure—affinity relationship (3D-QSAR) at the human 5-HT₇ receptor for comparison with similar studies at the highly homologous 5-HT_1A receptor. We report 35 new 5-SAT ligands, some with very high affinity (K_i ≤ 1 nM) and stereoselectivity at 5-HT₇ + or 5-HT_1A receptors, several with modest selectivity (up to 12-fold) for binding at 5-HT₇, and, several ligands with high selectivity (up to 40-fold) at the 5-HT_1A receptor. 3D-QSAR results indicate that steric extensions at the C(5)-position improve selectivity for the 5-HT₇ over 5-HT_1A receptor, while steric and hydrophobic extensions at the chiral C(2)-amino position impart 5-HT_1A selectivity. In silico receptor homology modeling studies, supplemented with molecular dynamics simulations and binding free energy calculations, were used to rationalize experimentally-determined receptor selectivity and stereoselective affinity results. The data from these studies indicate that the 5-SAT chemotype, previously shown to be safe and efficacious in rodent paradigms of neurodevelopmental and neuropsychiatric disorders, is amenable to structural modification to optimize affinity at serotonin 5-HT₇ vs. 5-HT_1A GPCRs, as may be required for successful clinical translation.     

Combined in situ hybridisation,northern blot analysis,and receptor binding studies in clones expressing different levels of the human 5-HT1A receptor

Abstract

In order to set up the technique of semi-quantitative in situ hybridisation to detect the serotonin receptor mRNA levels in brain tissue, a panel of three Swiss 3T3 cell clones (named clones 66, 53 and 47) expressing the human 5-HT_1A receptor at different densities were used as a model. The clones were generated by limiting dilution from pools of stably transfected cells. In addition membranes were prepared from each clone to perform receptor binding studies. Clones 66, 53, and 47 showed saturable binding for the agonist [³H]-8-OHDPAT, with receptor densities (B_max) of 227 ± 86, 548 ± 107 and 1505 ± 212 fmol/mg protein respectively, and with corresponding affinity constants (pKd) of 8.8 ± 0.1, 9.1 ± 0.1, and 9.1 ± 0.1 nM, respectively. Northern blot analysis using a specific probe for the 5-HT_IA receptor revealed the presence of a single 1.56 kilobase mRNA species in the 5-HT_1A receptor clones but not in control cells. In situ hybridisation studies were performed by measuring the 5-HT_1A receptor mRNA levels in these three 5-HT_1A transfectants using [³⁵S]αCTP labeled riboprobes (sense and anti-sense). The following rank order of receptor mRNA expression was found for clones 66, 53 and 47 respectively: 0.140 ± 0.001, 0.365 ± 0.045 and 0.835 ± 0.115 (relative optical density units). With the sense probe no specific labelling was observed. In conclusion, a positive correlation was found between receptor density (B_max) and receptor mRNA expression (semi-quantitative in situ hybridisation) using human 5-HT_1A receptor clones with different expression levels.     

Brainstem 5-Hydroxytrytamine1A Binding Sites are not Down-Regulated by Agonists which Induce Tolerance in the Rat: Myoclonus and other Serotonergic Behaviors

Abstract

To study the regulation of 5-HT_1A receptors in the brainstem, the region most relevant to the serotonin syndrome and to serotonin-responsive human myoclonic disorders, we chronically treated rats with various 5-HT_1A agonists and labeled 5-HT_1A sites with [³H]8-OH-DPAT. Daily injection for 30 consecutive days of 10 mg/kg ip 8-OH-DPAT (pre- and post-synaptic 5-HT_1A agonist) significantly decreased 8-OH-DPAT-evoked flat body posture, forelimb myoclonus, and hypothermia compared to chronic vehicle injection. There was no cross tolerance to 8-OH-DPAT in rats chronically injected with ipsapirone or buspirone (presynaptic 5-HT_1A agonists). However, none of the 5HT_1A agonists significantly altered B_max of brainstem 5-HT_1A binding sites. Chronic injection with other drugs such as 1-propranolol, (±) pindolol and spiperone (5-HT_1A and 5-HT₂ antagonists), methysergide (5-HT₁ and 5-HT₂ antagonist), and agonists and antagonists at various other 5-HT receptors also had no effect on binding parameters. These data demonstrate lack of cross-tolerance between pre- and post-synaptically acting 5-HT_1A agonists and absence of down-regulation of presynaptic 5-HT_1A sites at doses which induced tolerance of 5-HT_1A-mediated behaviors of the serotonin syndrome. They suggest changes in the post-synaptic cell rather than the receptor recognition site as the mechanism of tolerance.     

Membrane organization of the human serotonin1A receptor monitored by detergent insolubility using GFP fluorescence

Insolubility in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion for characterization of membrane domains. In view of the emerging role of membrane organization in the function of G-protein coupled receptors, we have examined detergent insolubility of the 5-HT_1A receptor in CHO cells using a novel GFP fluorescence approach developed by us. Using this approach, we have explored the membrane organization of the serotonin_1A receptor tagged to enhanced yellow fluorescent protein (5-HT_1AR-EYFP) stably expressed in CHO-K1 cells under conditions of varying detergent concentration, reduced membrane cholesterol and agonist stimulation. Our results show that a small yet significant fraction of the 5-HT_1A receptor exhibits detergent insolubility, which increases upon depletion of membrane cholesterol. Stimulation of 5-HT_1AR-EYFP by its endogenous ligand, serotonin, did not cause a significant change in the detergent insolubility of the receptor. Taken together, our results on detergent insolubility of 5-HT_1AR-EYFP provide new insights into the membrane organization of the 5-HT_1A receptor and could be relevant in the analysis of membrane organization of other G-protein coupled receptors.     

[3H]Spiroxatrine labels a serotonin1A-like site in the rat hippocampus

[³H]Spiroxatrine was examined as a potential ligand for the labeling of 5-HT_1A sites in the rat hippocampus. Analysis of the binding of [³H]spiroxatrine in the absence and presence of varying concentrations of three monoamine neurotransmitters revealed that serotonin (5-HT) had high affinity (IC₅₀= 20.7 nM for the [³H]spiroxatrine binding sites, consistent with the labeling of 5-HT₁ sites, while dopamine and norepinephrine had very low affinity (IC₅₀=57600 nM and >10⁻⁴ M respectively). Saturation studies of the binding of [³H]spiroxatrine revealed a single population of sites with a K_d=2.21 nM. Further pharmacologic characterization with the 5-HT_1A ligands 8-hydroxy-2-(di-n-propylamino) tetralin, ipsapirone, and WB4101 and the butyrophenone compounds spiperone and haloperidol gave results that were consistent with [³H]spiroxatrine labeling 5-HT_1A sites. This ligand produced stable, reproducible binding with a good ratio of specific to nonspecific binding. The binding of [³H]spiroxatrine was sensitive to GTP, suggesting that this ligand may act as an agonist. This was supported by the finding that spiroxatrine inhibits forskolin-stimulated adenylate cyclase activity (a proposed 5-HT_1A receptor model) in the rat hippocampus. Since [³H]spiroxatrine is structurally distinct from other currently available radioligands for the 5-HT_1A site, it should provide new information about the properties of this putative serotonergic receptor.     

The Human Serotonin 1A Receptor Expressed in Neuronal Cells: Toward a Native Environment for Neuronal Receptors

1. The serotonin_1A (5-HT_1A) receptor is an important representative of G-protein coupled family of receptors. It is the most extensively studied among the serotonin receptors, and appears to be involved in various behavioral and cognitive functions.2. We report here the pharmacological and functional characterization of the human serotonin_1A receptor stably expressed in HN2 cell line, which is a hybrid cell line between hippocampal cells and mouse neuroblastoma.3. Our results show that serotonin_1A receptors in HN2-5-HT_1AR cells display ligand-binding properties that closely mimic binding properties observed with native receptors. We further demonstrate that the differential discrimination of G-protein coupling by the specific agonist and antagonist, a hallmark of the native receptor, is maintained for the receptor in HN2-5-HT_1AR cells. Importantly, the serotonin_1A receptor in HN2-5-HT_1AR cells shows efficient downstream signalling by reducing cellular cyclic AMP levels.4. We conclude that serotonin_1A receptors expressed in HN2-5-HT_1AR cells represent a useful model system to study serotonin_1A receptor biology, and is a potential system for solubilization and purification of the receptor in native-like membrane environment.     

Crowding stress inhibits serotonin 1A receptor-mediated increases in corticotropin-releasing factor mRNA expression and adrenocorticotropin hormone secretion in the Gulf toadfish

Stimulation of the serotonin 1A (5-HT_1A) receptor subtype by 5-HT has been shown to result in an elevation in plasma corticosteroid levels in both mammals and several species of teleost fish, including the Gulf toadfish (Opsanus beta); however, in the case of teleost fish, it is not clearly known at which level of the hypothalamic–pituitary–interrenal axis the 5-HT_1A receptor is stimulated. Additionally, previous investigations have revealed that chronic elevations of plasma cortisol mediate changes in brain 5-HT_1A receptor mRNA and protein levels via the glucocorticoid receptor (GR); thus, we hypothesized that the function of centrally activated 5-HT_1A receptors is reduced or abolished as a result of chronically elevated plasma cortisol levels and that this response is GR mediated. Our results are the first to demonstrate that intravenous injection of the 5-HT_1A receptor agonist, 8-OH-DPAT, stimulates a significant increase in corticotropin-releasing factor (CRF) precursor mRNA expression in the hypothalamic region and the release of adrenocorticotropic hormone (ACTH) from the pituitary of teleost fish compared to saline-injected controls. We also provide evidence that cortisol, acting via GRs, attenuates the 5-HT_1A receptor-mediated secretion of both CRF and ACTH.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

Allosteric Inhibition of Serotonin 5-HT<Subscript>7</Subscript> Receptors by Zinc Ions

The allosteric regulation of G protein-coupled receptors (GPCRs) is a well-known phenomenon, but there are only a few examples of allosteric modulation within the metabotropic serotonergic receptor family. Recently, we described zinc non-competitive interactions toward agonist binding at serotonin 5-HT_1A receptors, in which biphasic effects, involving potentiation at sub-micromolar concentrations (10 μM) and inhibition at sub-millimolar concentrations (500 μM) of Zn²⁺ in radioligand binding assays, were consistent with both the agonist and antagonist-like effects of zinc ions observed in in vivo studies. Here, we showed new data demonstrating zinc allosteric inhibition of both agonist and antagonist binding at human recombinant 5-HT₇ receptors stably expressed in HEK293 cells as observed by radioligand binding studies as well as zinc neutral antagonism displayed by the concentration of 10 μM in the functional LANCE assay. The allosteric nature of the effect of Zn on 5-HT₇ receptors was confirmed (1) in saturation studies in which zinc inhibited the binding of potent orthosteric 5-HT₇ receptor radioligands, the agonist [³H]5-CT, and the two antagonists [³H]SB-269970 and [³H]mesulergine, showing ceiling effect and differences in the magnitude of negative cooperativity (α = 0.15, 0.06, and 0.25, respectively); (2) in competition experiments in which 500 μM of zinc inhibited all radioligand displacements by non-labeled orthosteric ligands (5-CT, SB-269970, and clozapine), and the most significant reduction in affinity was observed for the 5-CT agonist (4.9–16.7-fold) compared with both antagonists (1.4–3.9-fold); and (3) in kinetic experiments in which 500 μM zinc increased the dissociation rate constants for [³H]5-CT and [³H]mesulergine but not for [³H]SB-269970. Additionally, in the functional LANCE test using the constitutively active HEK293 cell line expressing the 5-HT₇ receptor, 10 μM zinc had features of neutral antagonism and increased the EC₅₀ value of the 5-CT agonist by a factor of 3.2. Overall, these results showed that zinc can act as a negative allosteric inhibitor of 5-HT₇ receptors. Given that the inhibiting effects of low concentrations of zinc in the functional assay represent the most likely direction of zinc activity under physiological conditions, among numerous zinc-regulated proteins, the 5-HT₇ receptor can be considered a serotonergic target for zinc modulation in the CNS.     

Ligand Binding Characteristics of the Human Serotonin<Subscript>1<Emphasis Type="Italic">A</Emphasis></Subscript> Receptor Heterologously Expressed in CHO Cells

The serotonin_1A (5-HT_1A) receptors are important members of the superfamily of seven transmembrane domain G-protein coupled receptors. They appear to be involved in various behavioral, cognitive and developmental functions. Mammalian cells in culture heterologously expressing membrane receptors represent convenient systems to address problems in receptor biology. We report here the pharmacological characterization of one of the first isolated clones of CHO cells stably expressing the human 5-HT_1A receptor using the selective agonist 8-OH-DPAT and antagonist p-MPPF. In addition, we demonstrate that agonist and antagonist binding to the receptor exhibit differential sensitivity to the non-hydrolyzable GTP analogue, GTP--S, as was observed earlier with the native receptor from bovine hippocampus. These results show that the human 5-HT_1A receptor expressed in CHO cells displays characteristic features found in the native receptor isolated from bovine hippocampus and promises to be a potentially useful system for future studies of the receptor.These authors have contributed equally to the work