A convenient synthesis of the muscarinic cholinergic antagonist (R)-[N-methyl-3 H]quinuclidinyl benzilate methiodide.

A useful synthesis of (R)-[N-methyl-3 H]quinuclidinyl benzilate methiodide is described with the product characterized by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), tritium nuclear magnetic resonance (NMR), and mass spectrometry (MS). Several methods are provided to purify the radioligand, and its storage and stability are also discussed.

Novel Stability-Indicating Chemometric-Assisted Spectrophotometric Methods for the Determination of Chlordiazepoxide and Clidinium Bromide in the Presence of Clidinium Bromide's Alkali-Induced Degradation Product.

Two simple and accurate chemometric-assisted spectrophotometric models were developed and validated for the simultaneous determination of chlordiazepoxide (CDZ) and clidinium bromide (CDB) in the presence of an alkali-induced degradation product of CDB in their pure and pharmaceutical formulation. Resolution was accomplished by using two multivariate calibration models, including principal component regression (PCR) and partial least-squares (PLS), applied to the UV spectra of the mixtures. Great improvement in the predictive abilities of these multivariate calibrations was observed. A calibration set was constructed and the best model used to predict the concentrations of the studied drugs. CDZ and CDB were analyzed with mean accuracies of 99.84 ± 1.41 and 99.81 ± 0.89% for CDZ and 99.56 ± 1.43 and 99.44 ± 1.41% for CDB using PLS and PCR models, respectively. The proposed models were validated and applied for the analysis of a commercial formulation and laboratory-prepared mixtures. The developed models were statistically compared with those of the official and reported methods with no significant differences observed. The models can be used for the routine analysis of both drugs in QC laboratories.

Graded activation and free energy landscapes of a muscarinic G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) recognize ligands of widely different efficacies, from inverse to partial and full agonists, which transduce cellular signals at differentiated levels. However, the mechanism of such graded activation remains unclear. Using the Gaussian accelerated molecular dynamics (GaMD) method that enables both unconstrained enhanced sampling and free energy calculation, we have performed extensive GaMD simulations (∼19 µs in total) to investigate structural dynamics of the M2 muscarinic GPCR that is bound by the full agonist iperoxo (IXO), the partial agonist arecoline (ARC), and the inverse agonist 3-quinuclidinyl-benzilate (QNB), in the presence or absence of the G-protein mimetic nanobody. In the receptor-nanobody complex, IXO binding leads to higher fluctuations in the protein-coupling interface than ARC, especially in the receptor transmembrane helix 5 (TM5), TM6, and TM7 intracellular domains that are essential elements for GPCR activation, but less flexibility in the receptor extracellular region due to stronger binding compared with ARC. Two different binding poses are revealed for ARC in the orthosteric pocket. Removal of the nanobody leads to GPCR deactivation that is characterized by inward movement of the TM6 intracellular end. Distinct low-energy intermediate conformational states are identified for the IXO- and ARC-bound M2 receptor. Both dissociation and binding of an orthosteric ligand are observed in a single all-atom GPCR simulation in the case of partial agonist ARC binding to the M2 receptor. This study demonstrates the applicability of GaMD for exploring free energy landscapes of large biomolecules and the simulations provide important insights into the GPCR functional mechanism.

Memetic algorithms for ligand expulsion from protein cavities.

Ligand diffusion through a protein interior is a fundamental process governing biological signaling and enzymatic catalysis. A complex topology of channels in proteins leads often to difficulties in modeling ligand escape pathways by classical molecular dynamics simulations. In this paper, two novel memetic methods for searching the exit paths and cavity space exploration are proposed: Memory Enhanced Random Acceleration (MERA) Molecular Dynamics (MD) and Immune Algorithm (IA). In MERA, a pheromone concept is introduced to optimize an expulsion force. In IA, hybrid learning protocols are exploited to predict ligand exit paths. They are tested on three protein channels with increasing complexity: M2 muscarinic G-protein-coupled receptor, enzyme nitrile hydratase, and heme-protein cytochrome P450cam. In these cases, the memetic methods outperform simulated annealing and random acceleration molecular dynamics. The proposed algorithms are general and appropriate in all problems where an accelerated transport of an object through a network of channels is studied.

Cholinergic antagonist 3-quinuclidinyl benzilate - Impact on learning and memory in Wistar rats.

3-Quinuclidinyl benzilate (QNB) represents a non-selective, competitive antagonist of cholinergic receptors, which has been previously used to generate cognitive deficits in animal models of neurodegenerative disorders. The aim of this study was evaluation of QNB potency for creation of cognitive impairment during the acquisition, consolidation and retrieval stages of learning and memory in rats. Male Wistar rats were subjected to a water maze task with hidden platform and a step-through passive avoidance task. The water maze test was carried out in two separate experiments focused on spatial learning (acquisition test) and long-term spatial memory (retrieval test). QNB doses (0.5, 1.0, 2.0 and 5.0 mg kg(-1)) were administered to rats intraperitoneally before training sessions (acquisition test) or before probe trial (retrieval test). A QNB dose of 2.0 mg kg(-1) was administered to rats in the passive avoidance task before training (acquisition test), immediately post-training (consolidation test) or 24h pre-retention (retrieval test). QNB significantly impaired the acquisition in the water maze at doses 0.5-5.0 mg kg(-1) as well as the acquisition of passive avoidance task. In contrast, consolidation and retrieval were not affected by QNB, indicating that QNB specifically affects the stage of acquisition.

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist.

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

Development of a homogeneous high-throughput live-cell G-protein-coupled receptor binding assay.

The measurement of ligand receptor binding parameters for G-protein-coupled receptors is indispensable in the drug discovery process. Traditional ligand receptor binding assays require scale-up of cells and membrane preparations, which is an expensive and time-consuming process. In this report, the authors describe the development of a homogeneous live-cell binding assay for GPCRs using a fluorophore-labeled nonpeptide ligand. The model assay used Cy3B-labeled telenzepine and Chinese hamster ovary cells expressing M1 muscarinic acetylcholine receptors. This homogeneous live-cell fluorescence binding assay format is superior to the traditional binding methods because it measures binding of a ligand to intact receptors on living cells. The assay requires no washing or separation steps, thereby allowing a real-time kinetic readout for the determination of ligand association and dissociation from the intact receptors. The results also suggest that miniaturization is feasible without compromising the data quality.

Recovery of oligomers and cooperativity when monomers of the M2 muscarinic cholinergic receptor are reconstituted into phospholipid vesicles.

FLAG- and HA-tagged M2 muscarinic receptors from coinfected Sf9 cells have been purified in digitonin-cholate and reconstituted into phospholipid vesicles. The purified receptor was predominantly monomeric: it showed no detectable coimmunoprecipitation; it migrated as a monomer during electrophoresis before or after cross-linking with bis(sulfosuccinimidyl)suberate; and it bound agonists and antagonists in a manner indicative of identical and mutually independent sites. Receptor cross-linked after reconstitution or after reconstitution and subsequent solubilization in digitonin-cholate migrated almost exclusively as a tetramer. The binding properties of the reconstituted receptor mimicked those reported previously for cardiac muscarinic receptors. The apparent capacity for N-[3H]methylscopolamine (NMS) was only 60% of that for [3H]quinuclidinylbenzilate (QNB), yet binding at saturating concentrations of [3H]QNB was inhibited fully and in a noncompetitive manner at comparatively low concentrations of unlabeled NMS. Reconstitution of the receptor with a saturating quantity of functional G proteins led to the appearance of three classes of sites for the agonist oxotremorine-M in assays with [3H]QNB; GMP-PNP caused an apparent interconversion from highest to lowest affinity and the concomitant emergence of a fourth class of intermediate affinity. All of the data can be described quantitatively in terms of cooperativity among four interacting sites, presumably within a tetramer; the effect of GMP-PNP can be accommodated as a shift in the distribution of tetramers between two states that differ in their cooperative properties. Monomers of the M2 receptor therefore can be assembled into tetramers with binding properties that closely resemble those of the muscarinic receptor in myocardial preparations.

Inhibition of ligand binding to G protein-coupled receptors by arachidonic acid.

Arachidonic acid (AA), released in response to muscarinic acetylcholine receptor (mAChR) stimulation, previously has been reported to function as a reversible feedback inhibitor of the mAChR. To determine if the effects of AA on binding to the mAChR are subtype specific and whether AA inhibits ligand binding to other G protein-coupled receptors (GPCRs), the effects of AA on ligand binding to the mAChR subtypes (M1, M2, M3, M4, and M5) and to the micro-opioid receptor, beta2-adrenergic receptor (beta2-AR), 5-hydroxytryptamine receptor (5-HTR), and nicotinic receptors were examined. AA was found to inhibit ligand binding to all mAChR subtypes, to the beta2-AR, the 5-HTR, and to the micro-opioid receptor. However, AA does not inhibit ligand binding to the nicotinic receptor, even at high concentrations of AA. Thus, AA inhibits several types of GPCRs, with 50% inhibition occurring at 3-25 MuM, whereas the nicotinic receptor, a non-GPCR, remains unaffected. Further research is needed to determine the mechanism by which AA inhibits GPCR function.

Monomers and oligomers of the M2 muscarinic cholinergic receptor purified from Sf9 cells.

G protein-coupled receptors are known to form oligomers. To probe the nature of such aggregates, as well as the role and prevalence of monomers, epitope-tagged forms of the M(2) muscarinic receptor have been isolated as oligomers and monomers from Sf9 cells. Membranes from cells coexpressing the c-Myc- and FLAG-tagged receptor were solubilized in digitonin-cholate, and the receptor was purified by successive passage through DEAE-Sepharose, the affinity resin 3-(2'-aminobenzhydryloxy)tropane (ABT)-Sepharose, and hydroxyapatite. Coimmunoprecipitation of the two epitopes indicated the presence of oligomers at each stage of the purification up to but not including the fraction eluted specifically from ABT-Sepharose. The affinity-purified receptor therefore appeared to be monomeric. The failure to detect coimmunoprecipitation was not due to an ineffective antibody, nor did the conditions of purification appear to promote disaggregation. Receptor at all stages of purification bound N-[(3)H]methylscopolamine and [(3)H]quinuclidinylbenzilate with high affinity, but the capacity of receptors that were not retained on ABT-Sepharose was only 4% of that expected from densitometry of western blots probed with an anti-M(2) antibody. Similarly low activity was found with oligomers isolated by successive passage of coexpressed receptor on anti-c-Myc and anti-FLAG immunoaffinity columns. M(2) muscarinic receptors therefore appear to coexist as active monomers and largely or wholly inactive oligomers in solubilized extracts of Sf9 cells. A different pattern emerged when coinfected cells were treated with quinuclidinylbenzilate prior to solubilization, in that ABT-purified receptors from those cells exhibited coimmunoprecipitation. Treatment with the antagonist therefore led to oligomers in which at least some of the constituent sites were active and were retained by ABT-Sepharose.

Capillary electrophoresis/mass spectrometry: a promising tool for the control of some physiologically hazardous compounds. I-derivatives of 3-quinuclidinol.

A method based on the coupling of capillary electrophoresis with mass spectrometry (CE/MS) was developed for the monitoring of 3-quinuclidinol and its four N-alkyl derivatives (methyl, ethyl, propyl and isopropyl derivatives). A fragmentation study (collision-induced dissociation of ions in an ion trap) and optimization of the ion optics set-up for CE/MS experiments using direct infusion of a methanolic solution of the standards into the mass spectrometer were carried out in advance. Molecular ions of all quaternary compounds and the quasi-molecular ion [M + H]+ of free 3-quinuclidinol prevail in the mass spectra. In the MS/MS of propyl and isopropyl derivatives, the elimination of the alkyl chain dominates, leading to the ion at m/z 128. The fragmentation of the other compounds is more complex. Previous CE separation of the mixture of isobaric propyl and isopropyl derivatives is necessary for their unambiguous identification. A 10 mM ammonium acetate buffer (pH 4.0) is the optimum running electrolyte, allowing the CE separation of methyl, ethyl, propyl and isopropyl derivatives. A 0.5% (v/v) solution of acetic acid in methanol provides sufficient detection sensitivity when used as the sheath liquid. Limits of detection of 0.1 ppm for 3-quinuclidinol and 0.05 ppm for quaternary derivatives were achieved under the optimum conditions. The optimized method was applied to the determination of 3-quinuclidinol and related quaternary derivatives spiked into a sample of pond water. The experimental set-up for CE/MS/MS was investigated, which strongly increases the identification capability of the technique.

Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors.

Muscarinic cholinergic receptors can appear to be more numerous when labeled by [(3)H]quinuclidinylbenzilate (QNB) than by N-[(3)H]methylscopolamine (NMS). The nature of the implied heterogeneity has been studied with M(2) receptors in detergent-solubilized extracts of porcine atria. The relative capacity for [(3)H]NMS and [(3)H]QNB was about 1 in digitonin-cholate, 0.56 in cholate-NaCl, and 0.44 in Lubrol-PX. Adding digitonin to extracts in cholate-NaCl increased the absolute capacity for both radioligands, and the relative capacity increased to near 1. The latency cannot be attributed to a chemically impure radioligand, instability of the receptor, an irreversible effect of NMS, or a failure to reach equilibrium. Binding at near-saturating concentrations of [(3)H]QNB in cholate-NaCl or Lubrol-PX was blocked fully by unlabeled NMS, which therefore appeared to inhibit noncompetitively at sites inaccessible to radiolabeled NMS. Such an effect is inconsistent with the notion of functionally distinct, noninterconverting, and mutually independent sites. Both the noncompetitive effect of NMS on [(3)H]QNB and the shortfall in capacity for [(3)H]NMS can be described quantitatively in terms of cooperative interactions within a receptor that is at least tetravalent; no comparable agreement is possible with a receptor that is only di- or trivalent. The M(2) muscarinic receptor therefore appears to comprise at least four interacting sites, presumably within a tetramer or larger array, and ligands appear to bind in a cooperative manner under at least some conditions.

The determination of a degradation product in clidinium bromide drug substance by capillary electrophoresis with indirect UV detection.

A capillary electrophoresis (CE) method utilizing indirect ultraviolet (UV) detection was developed for the determination of a non-UV absorbing degradation product, Ro 5-5172, in clidinium bromide drug substance. The electrophoresis buffer consisted of sodium phosphate and benzyltrimethylammonium bromide. Rinsing the capillary with sodium hydroxide followed by water then fresh capillary electrophoresis buffer was found to significantly improve the reproducibility of the migration times of the analytes. To further improve run-to-run reproducibility, an internal marker was used to account for differences in injection volumes and migration times between runs. The precision of the method was found to be less than 1% relative standard deviation for the migration time ratio and peak area ratio of Ro 5-5172 to the internal standard. The method was found to be linear for 0.05-1% Ro 5-5172 with respect to a 10 mg ml-1 sample preparation. The limit of detection was found to be less than 0.01% Ro 5-5172. Results obtained for the analysis of a clidinium bromide drug substance lot using this CE method and a thin layer chromatography method were compared and found to be in agreement.

Correction of the stereochemical assignment of the benzilic acid center in (R)-(-)-3-quinuclidinyl (S)-(+)-4-iodobenzilate [(R,S)-4-IQNB].

Radioiodinated (R)-quinuclidinyl-4-iodobenzilate (4IQNB) is a high affinity muscarinic antagonist which has been utilized for in vitro and in vivo assays, and for SPECT imaging in humans. 4IQNB exists in four different diastereomeric forms, since there are two asymmetric centers at the quinuclidinyl and benzilic acid centers. Based upon our in vivo studies, we have determined that the absolute stereochemistry previously assigned to the benzilic center was incorrect for the diastereomer that had been previously referred to as '(R)-quinuclidinyl-(R)-4-iodobenzilate' [(R,R)-4IQNB]. The correct designation for this diastereomer is '(R)-quinuclidinyl-(S)-4-iodobenzilate' [(R,S)-4IQNB].

In vivo autoradiography of radioiodinated (R)-3-quinuclidinyl (S)-4-iodobenzilate [(R, S)-IQNB] and (R)-3-quinuclidinyl (R)-4-iodobenzilate [(R,R)-IQNB]. Comparison of the radiolabelled products of a novel tributylstannyl precursor with those of the established triazene and exchange methods.

Radioiodinated (R,S)-IQNB and (R,R)-IQNB are prepared either from a triazene precursor or using an exchange reaction. In both cases the radiochemical yield is low. The product of the exchange reaction also suffers from having a fairly low specific activity. A new method for preparing radioiodinated (R,S)-IQNB and (R,R)-IQNB from a tributylstannyl precursor has recently been developed. This method is more convenient and much faster than the triazene and exchange methods, and it reliably results in a high radiochemical yield of a high specific activity product. In rat brain, the in vivo properties of the radioiodinated products of the tributylstannyl method are identical to those of the corresponding radioiodinated (R,S)-IQNB and (R,R)-IQNB prepared using the triazene and exchange methods. Dissection studies of selected brain regions show that at 3 h post injection (R,S)-[125I]IQNB prepared by all three methods have indistinguishable % dose g-1 values in all brain regions studied. Autoradiographic comparison of coronal slices through the anteroventral nucleus of the thalamus, through the hippocampus and through the pons at 2 h post injection shows that (R,S)-[125I]IQNB prepared by the triazene and tributylstannyl methods have indistinguishable patterns of binding.

An improved method for rapid and efficient radioiodination of iodine-123-IQNB.

UNLABELLED: The SPECT radioligand, 3-quinuclidinyl-4-[123I]iodobenzilate ([123I]IQNB), binds to muscarinic receptors and has generated interest as a potential agent for clinical SPECT. Unfortunately, cumbersome and inefficient radioiodination procedures have limited the practicality of [123I]IQNB SPECT imaging. METHODS: We report a rapid (5 min) and simple radioiodination procedure for preparing [123I]IQNB from a tri-n-butylstannyl precursor in a no-carrier-added reaction that yields high specific activity with radiochemical yield exceeding 60%. The radiochemical purity of the final product exceeds 95%. RESULTS: We have used this procedure to radioiodinate the four stereoisomers of [123I]IQNB. The procedure is highly reliable and reproducible. SPECT studies on a healthy human volunteer at 1, 2, 6 and 24 hr after injection of each of the four stereoisomers reveal expected differences in the kinetic and binding characteristics of the four stereoisomers. (R,S)-[123I]IQNB appears to be the SPECT agent of choice. CONCLUSION: Radioiodination of [123I]IQNB from our tri-n-butylstannyl precursor is simpler, more efficient and less expensive than previous techniques. The potential exists for a "kit" which would be practical in a typical clinical setting.

Formation of the functional complexes of m2 muscarinic acetylcholine receptors with GTP-binding regulatory proteins in solution.

Sixteen different detergents were studied for solubilization of functional complexes between m2 muscarinic acetylcholine receptors (mAChR) and guanine nucleotide-binding regulatory proteins (G proteins). More than 40% of solubilized mAChR retained their GTP-dependent high affinity for agonist binding after solubilization with sucrose monolaurate, whereas all other detergents studied gave considerably lower solubilization yields or caused the loss of the high affinity for agonist binding. The preformation of mAChR-G protein complexes in membranes revealed that a large excess of G proteins did not increase the portion of high-affinity binding sites, but caused GTP- and Mg2(+)-dependent inhibition of the binding of radioactive antagonists to mAChR. The optimization of detergent concentration and other experimental conditions revealed that up to 47% of the solubilized receptors indicated the GTP-dependent high affinity for agonist binding after mixing solubilized mAChR with purified G proteins in sucrose monolaurate in the presence of Mg2+ and carbachol. These results give the first clear proof of the formation of functional complexes between mAChR and G proteins in solution and indicate that GTP-dependent high-affinity agonist binding is connected to the direct interactions between mAChR and G proteins and that other membrane components are not necessary.

Muscarinic binding sites of the pig intravesical ureter.

1. Muscarinic receptors in the pig intravesical ureter were characterized by binding assays in which the muscarinic receptor antagonist, (-)-[3H]-quinuclidinyl benzylate ([3H]QNB) was used as radioligand. 2. The specific binding of [3H]-QNB (about 90% of the total binding, as defined with 10(-5) M unlabelled atropine) was dependent on protein concentration, saturable, and of high affinity (KD = 0.13 +/- 0.02 nM). 3. Displacement of [3H]-QNB specific binding by the M1-selective antagonist, pirenzepine, described a two-component curve, with a minor (17%) high affinity component (pKiH = 8.75), and a major (83%) low affinity one (pKiL = 6.34). The M3-preferential antagonists, hexa-hydro-sila-difenidol (HHSid) and p-fluoro-HHSiD (p-F-HHSiD) delineated also two sites, with pKiH of 8.91 and 8.57 and pKiL of 6.94 and 7.05, respectively. However, the M2-selective antagonists, 11-(2-(diethyl-amino)methyl-1-piperidinylacetyl)-5,11-dihydro-6H-p yrido-(2,3-b)- (1,4)-benzodiazepin-6-one (AF-DX 116, pKi = 6.72) and methoctramine (pKi = 8.34), as well as the M4-selective antagonists, tropicamide (pKi = 7.15) and himbacine (pKi = 8.65) fitted best to a single population of sites. Moreover, 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP), a muscarinic antagonist that discriminates the M1 and M3 versus the M2 subtypes, also delineated one site (pKi = 8.36). 4. The antagonist profile clearly indicates the existence of an M2 population in the porcine intravesical ureter. In addition, the presence of a minor non-M2 population, which may be formed by a mixture of several muscarinic subtypes (i.e. M1, M3 and/or M4) can not be discounted. 5. The present work confirms the results obtained in previous functional studies where the stimulation of muscarinic receptors by carbachol evoked the contraction of the pig isolated intravesical ureter.

Effect of phospholipid hydrolysis by phospholipase A2 on the kinetics of antagonist binding to cardiac muscarinic receptors.

Activation of phospholipases during prolonged myocardial ischemia could contribute to the functional derangement of myocardial cells by altering the phospholipid environment of a number of membrane bound proteins including receptors. The present study examined the kinetics of muscarinic receptor antagonist [3H]quinuclidinyl benzilate binding ([3H]QNB) to muscarinic receptors of highly purified sarcolemmal membranes under control conditions and after treatment with phospholipase A2 (PLA2; EC 3.1.1.4). Initial binding rates of QNB exhibited saturation kinetics, when plotted against the ligand concentration in control and PLA2 treated sarcolemmal membranes. This kinetic behaviour of QNB-binding is consistent with at least a two step binding mechanism. According to this two step binding hypothesis an unstable intermediate receptor-QNB complex (R*QNB) forms rapidly, and this form undergoes a slow conversion to the high affinity ligand-receptor complex R-QNB. The Michaelis constant Km of R-QNB formation was 1.8 nM, whereas the dissociation constant Kd obtained from equilibrium measurements was 0.062 nM. After 5 min exposure of sarcolemmal membranes to PLA2QNB binding capacity (Bmax) was reduced by 62%, and the affinity of the remaining receptor sites was decreased by 47% (Kd = 0.116 nM). This PLA2-induced increase of Kd was accompanied by a corresponding increase of Km, whereas the rate constants k2 and k-2 of the hypothetical slow conversion step (second reaction step) remained unchanged. These results suggest that binding of QNB to cardiac muscarinic receptors induces a transition in the receptor-ligand configuration, which is necessary for the formation of the final high affinity R-QNB complex. PLA2-induced changes of the lipid environment result in the inability of a part of the receptor population to undergo this transition, thereby inhibiting high affinity QNB-binding.