Evolutionary trace analysis of ionotropic glutamate receptor sequences and modeling the interactions of agonists with different NMDA receptor subunits

The ionotropic N-methyl-d-aspartate (NMDA) receptor is of importance in neuronal development, functioning, and degeneration in the mammalian central nervous system. The functional NMDA receptor is a heterotetramer comprising two NR1 and two NR2 or NR3 subunits. We have carried out evolutionary trace (ET) analysis of forty ionotropic glutamate receptor (IGRs) sequences to identify and characterize the residues forming the binding socket. We have also modeled the ligand binding core (S1S2) of NMDA receptor subunits using the recently available crystal structure of NR1 subunit ligand binding core which shares ~40% homology with other NMDA receptor subunits. A short molecular dynamics simulation of the glycine-bound form of wild-type and double-mutated (D481N; K483Q) NR1 subunit structure shows considerable RMSD at the hinge region of S1S2 segment, where pore forming transmembrane helices are located in the native receptor. It is suggested that the disruption of domain closure could affect ion-channel activation and thereby lead to perturbations in normal animal behavior. In conclusion, we identified the amino acids that form the ligand-binding pocket in many ionotropic glutamate receptors and studied their hydrogen bonded and nonbonded interaction patterns. Finally, the disruption in the S1S2 domain conformation (of NR1 subunit- crystal structure) has been studied with a short molecular dynamics simulation and correlated with some experimental observations.Figure The figure shows the binding mechanism of glutamate with NR2B subunit of the NMDA receptor. Glutamate is shown in cpk, hydrogen bonds in dotted lines and amino acids in blue. The amino acids shown here are within a 4-Å radius of the ligand (glutamate)     

Stimulation of human formyl peptide receptors by calpain inhibitors: homology modeling of receptors and ligand docking simulation

Calpain inhibitors, including peptide aldehydes (N-acetyl-Leu-Leu-Nle-CHO and N-acetyl-Leu-Leu-Met-CHO) and α-mercapto-acrylic acid derivatives (PD150606 and PD151746), have been shown to stimulate phagocyte functions via activation of human formyl peptide receptor (hFPR) and/or hFPR-like 1 (hFPRL1). Using the homology modeling of the receptors and the ligand docking simulation, here we show that these calpain inhibitors could bind to the putative N-formyl-Met-Leu-Phe (fMLF) binding site on hFPR and/or hFPRL1. The studies with HEK-293 cells stably expressing hFPR or hFPRL1 showed that the concentrations of calpain inhibitors required to induce an increase in cytoplasmic free Ca²⁺ ([Ca²⁺]_i) was much higher (>100 folds) than those of fMLF and Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm). HEK-293 cells expressing hFPR or hFPRL1 with the mutated fMLF binding site never exhibited the [Ca²⁺]_i response to calpain inhibitors. When the optimal concentrations of each stimulus were used, pretreatment of cells with fMLF or WKYMVm abolished an increase in [Ca²⁺]_i induced by calpain inhibitors as well as the same stimulus, whereas pretreatment of cells with calpain inhibitors significantly suppressed, but never abolished, the [Ca²⁺]_i response induced by fMLF or WKYMVm, suggesting that the binding affinity of the inhibitors to the putative fMLF binding site may be lower than that of fMLF or WKYMVm.     

G protein-coupled adenosine (P1) and P2Y receptors: ligand design and receptor interactions

The medicinal chemistry and pharmacology of the four subtypes of adenosine receptors (ARs) and the eight subtypes of P2Y receptors (P2YRs, activated by a range of purine and pyrimidine mono- and dinucleotides) has recently advanced significantly leading to selective ligands. X-ray crystallographic structures of both agonist- and antagonist-bound forms of the A(2A)AR have provided unprecedented three-dimensional detail concerning molecular recognition in the binding site and the conformational changes in receptor activation. It is apparent that this ubiquitous cell signaling system has implications for understanding and treating many diseases. ATP and other nucleotides are readily released from intracellular sources under conditions of injury and organ stress, such as hypoxia, ischemia, or mechanical stress, and through channels and vesicular release. Adenosine may be generated extracellularly or by cellular release. Therefore, depending on pathophysiological factors, in a given tissue, there is often a tonic activation of one or more of the ARs or P2YRs that can be modulated by exogenous agents for a beneficial effect. Thus, this field has provided fertile ground for pharmaceutical development, leading to clinical trials of selective receptor ligands as imaging agents or for conditions including cardiac arrhythmias, ischemia/reperfusion injury, diabetes, pain, thrombosis, Parkinson's disease, rheumatoid arthritis, psoriasis, dry eye disease, pulmonary diseases such as cystic fibrosis, glaucoma, cancer, chronic hepatitis C, and other diseases.     

Electric field interactions in pairs of electric fish: modeling and mimicking naturalistic inputs

Weakly electric fish acquire information about their surroundings by detecting and interpreting the spatial and temporal patterns of electric potential across their skin, caused by perturbations in a self-generated, oscillating electric field. Computational and experimental studies have focused on understanding the electric images due to simple, passive objects. The present study considers electric images of a conspecific fish. It is known that the electric fields of two fish interact to produce beats with spatially varying profiles of amplitude and phase. Such patterns have been shown to be critical for electrosensory-mediated behaviours, such as the jamming avoidance response, but they have yet to be well described. We have created a biophysically realistic model of a wave-type weakly electric fish by using a genetic algorithm to calibrate the parameters to the electric field of a real fish. We use the model to study a pair of fish and compute the electric images of one fish onto the other at three representative phases within a beat cycle. Analysis of the images reveals rostral/caudal and ipsilateral/contralateral patterns of amplitude and phase that have implications for localization of conspecifics (both position and orientation) and communication between conspecifics. We then show how the common stimulation paradigm used to mimic a conspecific during in vivo electrophysiological experiments, based on a transverse arrangement of two electrodes, can be improved in order to more accurately reflect the important qualitative features of naturalistic inputs, as revealed by our model.     

Expression and function of Toll-like receptors in eosinophils: activation by Toll-like receptor 7 ligand

We investigated the expression of a panel of Toll-like receptors (TLRs) and their functions in human eosinophils. Eosinophils constitutively expressed TLR1, TLR4, TLR7, TLR9, and TLR10 mRNAs (TLR4 greater than TLR1, TLR7, TLR9, and TLR10 greater than TLR6). In contrast, neutrophils expressed a larger variety of TLR mRNAs (TLR1, TLR2, TLR4, TLR6, TLR8 greater than TLR5, TLR9, and TLR10 greater than TLR7). Although the expression levels in eosinophils were generally less prominent compared with those in neutrophils, eosinophils expressed a higher level of TLR7. Furthermore, among various TLR ligands (S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-Cys-Ser-(Lys)(4), poly(I:C), LPS, R-848, and CpG DNA), only R-848, a ligand of TLR7 and TLR8, regulated adhesion molecule (CD11b and L-selectin) expression, prolonged survival, and induced superoxide generation in eosinophils. Stimulation of eosinophils by R-848 led to p38 mitogen-activated protein kinase activation, and SB203580, a p38 mitogen-activated protein kinase inhibitor, almost completely attenuated R-848-induced superoxide generation. Although TLR8 mRNA expression was hardly detectable in freshly isolated eosinophils, mRNA expression of TLR8 as well as TLR7 was exclusively up-regulated by IFN-gamma but not by either IL-4 or IL-5. The up-regulation of the TLRs by IFN-gamma had potentially functional significance: the extent of R-848-induced modulation of adhesion molecule expression was significantly greater in cells treated with IFN-gamma compared with untreated cells. Although the natural ligands for TLR7 and TLR8 have not yet been identified, our results suggest that eosinophil TLR7/8 systems represent a potentially important mechanism of a host-defensive role against viral infection and mechanism linking exacerbation of allergic inflammation and viral infection.     

Binding site and interlobe interactions of the ionotropic glutamate receptor GluK3 ligand binding domain revealed by high resolution crystal structure in complex with (S)-glutamate

Ionotropic glutamate receptors (iGluRs) are involved in excitatory signal transmission throughout the central nervous system and their malfunction is associated with various health disorders. GluK3 is a subunit of iGluRs, belonging to the subfamily of kainate receptors (GluK1–5). Several crystal structures of GluK1 and GluK2 ligand binding domains have been determined in complex with agonists and antagonists. However, little is known about the molecular mechanisms underlying GluK3 ligand binding properties and no compounds displaying reasonable selectivity towards GluK3 are available today. Here, we present the first X-ray crystal structure of the ligand binding domain of GluK3 in complex with glutamate, determined to 1.6 Å resolution. The structure reveals a conserved glutamate binding mode, characteristic for iGluRs, and a water molecule network in the glutamate binding site similar to that seen in GluK1. In GluK3, a slightly lower degree of domain closure around glutamate is observed compared to most other kainate receptor structures with glutamate. The volume of the GluK3 glutamate binding cavity was found to be of intermediate size between those of GluK1 and GluK2. The residues in GluK3 contributing to the subfamily differences in the binding sites are primarily: Thr520, Ala691, Asn722, Leu736 and Thr742. The GluK3 ligand binding domain seems to be less stabilized through interlobe interactions than GluK1 and this may contribute to the faster desensitization kinetics of GluK3.     

Characterization of low density lipoprotein receptor ligand interactions by fluorescence resonance energy transfer

The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. A soluble low density lipoprotein receptor (sLDLR) and a tryptophan (Trp)-deficient variant human apolipoprotein E3 (apoE3) N-terminal domain (NT) were used in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine (AEDANS), and the protein was complexed with lipid. Incubation of sLDLR with AEDANS-Trp-null apoE3-NT dimyristoylphosphatidylcholine (DMPC) disks, but not lipid-free AEDANS-apoE, induced an enhancement in AEDANS fluorescence emission intensity (excitation, 280 nm) consistent with intermolecular energy transfer from excited Trp in sLDLR to receptor-bound apoE. Ligand binding to sLDLR required calcium and was saturable. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low density lipoprotein. Fluorescence changes in this system reflected pH-dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Intermolecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for the characterization of the myriad molecular interactions ascribed to this family of receptor.     

Novel insights into GPCR-peptide interactions: mutations in extracellular loop 1, ligand backbone methylations and molecular modeling of neurotensin receptor 1

Investigating prototypical interactions between NT(8-13) and the human neurotensin receptor 1 (hNTR1), we created a receptor-ligand model that was validated by site-directed mutagenesis and structure-activity relationship studies. Stabilization of the extracellular loop 1 (EL1) by pi-stacking clusters proved to be important for agonist binding when substitution of six conserved amino acids by alanine resulted in an agonist specific loss of maximal binding capacity. In agreement with our modeling studies, EL1 seems to adopt a clamp-type border area controlling the shape of the binding site crevice. Employing chemically manipulated peptide analogs as molecular probes, the impact of backbone modifications on receptor-ligand interaction, especially the influence on ligand conformation, was examined in binding studies and explained by in silico analysis.     

New insights into the GABA(A) receptor structure and orthosteric ligand binding: receptor modeling guided by experimental data

GABA(A) receptors (GABA(A)Rs) are ligand gated chloride ion channels that mediate overall inhibitory signaling in the CNS. A detailed understanding of their structure is important to gain insights in, e.g., ligand binding and functional properties of this pharmaceutically important target. Homology modeling is a necessary tool in this regard because experimentally determined structures are lacking. Here we present an exhaustive approach for creating a high quality model of the α(1)β(2)γ(2) subtype of the GABA(A)R ligand binding domain, and we demonstrate its usefulness in understanding details of orthosteric ligand binding. The model was constructed by using multiple templates and by incorporation of knowledge from biochemical/pharmacological experiments. It was validated on the basis of objective energy functions, its ability to account for available residue specific information, and its stability in molecular dynamics (MD) compared with that of the two homologous crystal structures. We then combined the model with extensive structure-activity relationships available from two homologous series of orthosteric GABA(A)R antagonists to create a detailed hypothesis for their binding modes. Excellent agreement with key experimental data was found, including the ability of the model to accommodate and explain a previously developed pharmacophore model. A coupling to agonist binding was thereby established and discussed in relation to activation mechanisms. Our results highlight the importance of critical evaluation and optimization of each step in the homology modeling process. The approach taken here can greatly aid in increasing the understanding of GABA(A)Rs and related receptors where structural insight is limited and reliable models are difficult to obtain.     

Role of the extracellular loops of G protein-coupled receptors in ligand recognition: a molecular modeling study of the human P2Y1 receptor

The P2Y1 receptor is a G protein-coupled receptor (GPCR) and is stimulated by extracellular ADP and ATP. Site-directed mutagenesis of the three extracellular loops (ELs) of the human P2Y1 receptor indicates the existence of two essential disulfide bridges (Cys124 in EL1 and Cys202 in EL2; Cys42 in the N-terminal segment and Cys296 in EL3) and several specific ionic and H-bonding interactions (involving Glu209 and Arg287). Through molecular modeling and molecular dynamics simulations, an energetically sound conformational hypothesis for the receptor has been calculated that includes transmembrane (TM) domains (using the electron density map of rhodopsin as a template), extracellular loops, and a truncated N-terminal region. ATP may be docked in the receptor, both within the previously defined TM cleft and within two other regions of the receptor, termed meta-binding sites, defined by the extracellular loops. The first meta-binding site is located outside of the TM bundle, between EL2 and EL3, and the second higher energy site is positioned immediately underneath EL2. Binding at both the principal TM binding site and the lower energy meta-binding sites potentially affects the observed ligand potency. In meta-binding site I, the side chain of Glu209 (EL2) is within hydrogen-bonding distance (2.8 A) of the ribose O3', and Arg287 (EL3) coordinates both alpha- and beta-phosphates of the triphosphate chain, consistent with the insensitivity in potency of the 5'-monophosphate agonist, HT-AMP, to mutation of Arg287 to Lys. Moreover, the selective reduction in potency of 3'NH2-ATP in activating the E209R mutant receptor is consistent with the hypothesis of direct contact between EL2 and nucleotide ligands. Our findings support ATP binding to at least two distinct domains of the P2Y1 receptor, both outside and within the TM core. The two disulfide bridges present in the human P2Y1 receptor play a major role in the structure and stability of the receptor, to constrain the loops within the receptor, specifically stretching the EL2 over the opening of the TM cleft and thus defining the path of access to the binding site.     

Cation-pi interactions in ligand recognition by serotonergic (5-HT3A) and nicotinic acetylcholine receptors: the anomalous binding properties of nicotine

A series of tryptophan analogues has been introduced into the binding site regions of two ion channels, the ligand-gated nicotinic acetylcholine and serotonin 5-HT(3A) receptors, using unnatural amino acid mutagenesis and heterologous expression in Xenopus oocytes. A cation-pi interaction between serotonin and Trp183 of the serotonin channel 5-HT(3A)R is identified for the first time, precisely locating the ligand-binding site of this receptor. The energetic contribution of the observed cation-pi interaction between a tryptophan and the primary ammonium ion of serotonin is estimated to be approximately 4 kcal/mol, while the comparable interaction with the quaternary ammonium of acetylcholine is approximately 2 kcal/mol. The binding mode of nicotine to the nicotinic receptor of mouse muscle is examined by the same technique and found to differ significantly from that of the natural agonist, acetylcholine.     

Regulation of mucosal dendritic cell function by receptor activator of NF-kappa B (RANK)/RANK ligand interactions: impact on tolerance induction

The mucosal immune system is uniquely equipped to discriminate between potentially invasive pathogens and innocuous food proteins. While the mechanisms responsible for induction of mucosal immunity vs tolerance are not yet fully delineated, recent studies have highlighted mucosal dendritic cells (DC) as being important in determining the fate of orally administered Ag. To further investigate the DC:T cell signals involved in regulating the homeostatic balance between mucosal immunity and tolerance, we have examined the expression and function of the TNFR family member receptor activator of NF-kappaB (RANK) and its cognate ligand, RANKL, in vitro and in vivo. Our data show that although DC isolated from mucosal lymphoid tissues expressed similar levels of surface RANK compared with DC isolated from peripheral lymphoid tissues, DC from the distinct anatomical sites displayed differential responsiveness to RANK engagement with soluble RANKL. Whereas splenic DC responded to RANKL stimulation with elevated IL-12 p40 mRNA expression, Peyer's patch DC instead preferentially displayed increased IL-10 mRNA expression. Our data also show that the in vivo functional capacity of mucosal DC can be modulated by RANKL. Treatment with RANKL in vivo at the time of oral administration of soluble OVA enhanced the induction of tolerance in two different mouse models. These studies underscore the functional differences between mucosal and peripheral DC and highlight a novel role for RANK/RANKL interactions during the induction of mucosal immune responses.     

Fermentation at non-conventional conditions in food- and bio-sciences by the application of advanced processing technologies

The interest in improving the yield and productivity values of relevant microbial fermentations is an increasingly important issue for the scientific community. Therefore, several strategies have been tested for the stimulation of microbial growth and manipulation of their metabolic behavior. One promising approach involves the performance of fermentative processes during non-conventional conditions, which includes high pressure (HP), electric fields (EF) and ultrasound (US). These advanced technologies are usually applied for microbial inactivation in the context of food processing. However, the approach described in this study focuses on the use of these technologies at sub-lethal levels, since the aim is microbial growth and fermentation under these stress conditions. During these sub-lethal conditions, microbial strains develop specific genetic, physiologic and metabolic stress responses, possibly leading to fermentation products and processes with novel characteristics. In some cases, these modifications can represent considerable improvements, such as increased yields, productivities and fermentation rates, lower accumulation of by-products and/or production of different compounds. Although several studies report the successful application of these technologies during the fermentation processes, information on this subject is still scarce and poorly understood. For that reason, the present review paper intends to assemble and discuss the main findings reported in the literature to date, and aims to stimulate interest and encourage further developments in this field.     

Feed-forward signaling by membrane-bound ligand receptor circuit: the case of NOTCH DELTA-like 4 ligand in endothelial cells

The DELTA like-4 ligand (DLL4) belongs to the highly conserved NOTCH family and is specifically expressed in the endothelium. DLL4 regulates crucial processes in vascular growth, including endothelial cell (EC) sprouting and arterial specification. Its expression is increased by VEGF-A. In the present study, we show that VEGF-induced DLL4 expression depends on NOTCH activation. VEGF-induced DLL4 expression was prevented by the blockage of NOTCH signaling with γ-secretase or ADAM inhibitors in human cardiac microvascular ECs. Similar to VEGF-A, recombinant DLL4 itself stimulated NOTCH signaling and resulted in up-regulation of DLL4, suggesting a positive feed-forward mechanism. These effects were abrogated by NOTCH inhibitors but not by inhibition of VEGF signaling. NOTCH activation alone suffices to induce DLL4 expression as illustrated by the positive effect of NOTCH intracellular domain (NICD)-1 or -4 overexpression. To discriminate between NICD/RBP-Jκ and FOXC2-regulated DLL4 expression, DLL4 promoter activity was assessed in promoter deletion experiments. NICD induced promoter activity was dependent on RBP-Jκ site but independent of the FOXC2 binding site. Accordingly, constitutively active FOXC2 did not affect DLL4 expression. The notion that the positive feed-forward mechanism might propagate NOTCH activation to neighboring ECs was supported by our observation that DLL4-eGFP-transfected ECs induced DLL4 expression in nontransfected cells in their vicinity. In summary, our data provide evidence for a mechanism by which VEGF or ligand-induced NOTCH signaling up-regulates DLL4 through a positive feed-forward mechanism. By this mechanism, DLL4 could propagate its own expression and enable synchronization of NOTCH expression and signaling between ECs.     

Theoretical considerations and modeling of chemical inactivation of microorganisms: Inactivation of Giardia Cysts by free chlorine

Chemical inactivation of microorganisms is a common process widely employed in many fields such as in treatment of water, preservation in food industry and antimicrobial treatments in healthcare. For economy of applications and efficiency of treatment establishment the minimum dosage of breakpoint in the chemical application becomes essential. Even though experimental investigations have been extensive, theoretical understanding of such processes are demanding. Commonly employed theoretical analyses for the inactivation of microorganisms and depletion of chemicals include kinetics expressing the rates of depletion of chemical and microorganisms. The terms chemical demand (x) and specific disinfectant demand (α) are often used in theoretical modeling of inactivation. The value of specific disinfectant demand (α) has always been assumed to be a constant in these models. Intracellular concentration built up within the cells of the microorganisms during inactivation could lead to possible weakening effects of microorganisms thereby requiring lower doses as disinfection proceeds makes the assumption of constant α inaccurate. Model equations are formulated based on these observations co-relating the parameters α and x with a progressive inactivation (N/N₀). The chemical concentration (C) is also presented in terms of the inactivation time (t) and the survival ratio (N/N₀) for given pH and temperature conditions. The model is examined using experimentally verified Ct data of Giardia Cysts/chlorine system. The respective values of x for different survival ratios were evaluated from the data using MatLab software. Proposed model correlating for the disinfectant demand (x) with the survival ratio (N/N₀) fits satisfactorily with those evaluated from data. The rate constants for different pH and temperature conditions are evaluated which showed compatibility with the Arrhenius model. The dependence of frequency factors with pH indicated compatibility with accepted models. The Ct values regenerated with the kinetic data shows a very accurate fit with published data.     

The anticonvulsant effects of DADLE are primarily mediated by activation of delta opioid receptors: interactions between delta and mu receptor antagonists

Dose-response comparisons of the ability of the selective delta antagonist ICI 154,129 (12.5-50 nmol), the nonselective antagonist naloxone (29-290 nmol), and the irreversible selective mu antagonist beta-fNA (1.3-21 nmol) to alter the threshold response to DADLE or etorphine was studied in the rat flurothyl seizure test. DADLE (35 nmol, i.c.v.) and etorphine (122 nmol/kg, s.c.) both caused increases in seizure threshold which were differentially antagonized by pretreatment (i.c.v.) with the respective antagonists. For DADLE, only ICI 154,129 and naloxone produced a dose-related blockade of the increase in seizure threshold, with ICI 154,129 being more potent than naloxone. In contrast, the anticonvulsant action of etorphine was not antagonized by ICI 154,129 (50 nmol), but was blocked by a low dose of naloxone (29 nmol) or beta-fNA (21 nmol). In addition, prior occupancy of mu-sites with beta-fNA (21 nmol) significantly diminished the abilities of either ICI 154, 129 (50 nmol) or naloxone (290 nmol) to antagonize the anticonvulsant action of DADLE. The results of this study demonstrated that the effects of DADLE to increase seizure threshold in the rat were primarily mediated by activation of a delta-opioid receptor system. Furthermore, evidence has been provided for a functional interaction between delta and mu receptors in the opioid regulation of seizure threshold.     

Characterization of low affinity Fcγ receptor biotinylation under controlled reaction conditions by mass spectrometry and ligand binding analysis

Chemical protein biotinylation and streptavidin or anti‐biotin‐based capture is regularly used for proteins as a more controlled alternative to direct coupling of the protein on a biosensor surface. On biotinylation an interaction site of interest may be blocked by the biotin groups, diminishing apparent activity of the protein. Minimal biotinylation can circumvent the loss of apparent activity, but still a binding site of interest can be blocked when labeling an amino acid involved in the binding. Here, we describe reaction condition optimization studies for minimal labeling. We have chosen low affinity Fcγ receptors as model compounds as these proteins contain many lysines in their active binding site and as such provide an interesting system for a minimal labeling approach. We were able to identify the most critical parameters (protein:biotin ratio and incubation pH) for a minimal labeling approach in which the proteins of choice remain most active toward analyte binding. Localization of biotinylation by mass spectrometric peptide mapping on minimally labeled material was correlated to protein activity in binding assays. We show that only aiming at minimal labeling is not sufficient to maintain an active protein. Careful fine‐tuning of critical parameters is important to reduce biotinylation in a protein binding site.     

Study of ligand-receptor interactions by fluorescence correlation spectroscopy with different fluorophores: evidence that the homopentameric 5-hydroxytryptamine type 3As receptor binds only one ligand.

The 5-hydroxytryptamine receptor of type 3 was investigated by fluorescence correlation spectroscopy (FCS). Binding constants of fluorescently labeled ligands, the stoichiometry, and the mass of the receptor are readily accessible by this technique, while the duration of measurement is on the order of seconds to minutes. The receptor antagonist 1,2,3, 9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-aminopropyl)- 4H-carbazol-4-one (GR-H) was labeled with the fluorophores rhodamine 6G, fluorescein, N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl], and the cyanine dye Cy5. These labels cover a large part of the visible electromagnetic spectrum. It is shown that the photophysical and chemical properties have a direct influence on the measurement quality (duration of measurement, signal-to-noise ratio) and the ligand-receptor interactions (dissociation constants), respectively. This makes it necessary to choose a suitable label or a combination of labels for receptor studies. The affinities of the fluorescently labeled ligands determined by FCS were virtually identical to the values obtained by radioligand binding experiments. Moreover, the dissociation constant of a nonfluorescent receptor ligand was determined successfully by an FCS competition assay. The experimental results showed that only one antagonist binds to the receptor, in agreement with measurements previously published [Tairi et al. (1998) Biochemistry 37, 15850-15864].     

Novel drug interactions at D(2) dopamine receptors: modulation of [3H]quinpirole binding by monoamine oxidase inhibitors

D(2) dopamine receptors are the principal target of drugs used to treat schizophrenia and Parkinson's disease. Recent findings suggest novel drug interactions at D(2) receptors, specifically interactions of monoamine oxidase inhibitors (MAOIs) at a novel binding site that modulates the binding of [3H]quinpirole to the D(2) receptor. That MAOIs inhibit [3H]quinpirole binding challenges the traditional understanding of ligand interactions at dopamine receptors and may shed light on the mechanism of behavioral sensitization to psychostimulants and the pharmacology and toxicity of MAOIs.