Mutagenesis of the GABA(A) receptor alpha1 subunit reveals a domain that affects sensitivity to GABA and benzodiazepine-site ligands

We have mutated several amino acids in the region of the GABA(A) receptor alpha1 subunit predicted to form a small extracellular loop between transmembrane domains two and three to investigate its possible role in ligand sensitivity. The mutations were S275T, L276A, P277A, V279A, A280S and Y281F. Mutant alpha1 subunits were co-expressed with beta2 and gamma2 subunits in tsA201 cells or Xenopus oocytes. Binding studies revealed that the only mutation that significantly affected [3H]Ro15-4513 binding was the V279A substitution which reduced the affinity for this ligand. Electrophysiological examination of mutant receptors revealed that L276A, P277A and V279A displayed rightward shifts of their GABA concentration-response curves, the largest occurring with the L276A mutant. The impact of these mutations on allosteric modulation by benzodiazepine-site ligands was examined. V279A reduced the potency of both flunitrazepam and Ro15-4513 but, in each case, their efficacy was enhanced. A280S resulted in a decrease in flunitrazepam efficacy without affecting its potency. Additionally, P277A and A280S resulted in Ro15-4513 losing its inverse agonist effect at these receptors. These results suggest that a domain within this small extracellular loop between TMII-TMIII plays a role in determining the sensitivity of GABA(A) receptors to both GABA and benzodiazepine-site ligands.     

On high- and low-affinity agonist sites in GABAA receptors

GABAA receptors are activated via low-affinity binding sites for the agonists GABA or muscimol. Evidence has been provided that the amino acid residue alpha 1F64 located at the beta2(+)/alpha1(-) subunit interface forms part of this binding site. In radioactive ligand binding studies the agonist [3H]muscimol has been found to interact with the receptor via a high-affinity binding site. This site has been interpreted as a conformational variant of the low-affinity site. Alternatively, the high-affinity binding site has been located to the alpha1(+)/beta2(-) interface and the homologous residue to alpha 1F64, beta 2Y62 has been proposed to constitute an important part of this site. Here we investigated the effect of the point mutation alpha 1F64L and the homologous mutation beta 2Y62L on agonist and antagonist binding and functional properties in alpha 1 beta 2 gamma 2 GABAA receptors. While the mutation in the alpha1 subunit had drastic consequences on all studied properties, including desensitization, the mutation in the beta2 subunit had little consequence. Our observations are relevant for the relative location of high- and low-affinity agonist sites in GABAA receptors.     

GABA(A) receptors in aging and Alzheimer's disease

In this article we present a comprehensive review of relevant research and reports on the GABA_A receptor in the aged and Alzheimer's disease (AD) brain. In comparison to glutamatergic and cholinergic systems, the GABAergic system is relatively spared in AD, but the precise mechanisms underlying differential vulnerability are not well understood. Using several methods, investigations demonstrate that despite resistance of the GABAergic system to neurodegeneration, particular subunits of the GABA_A receptor are altered with age and AD, which can induce compensatory increases in GABA_A receptor subunits within surrounding cells. We conclude that although aging- and disease-related changes in GABA_A receptor subunits may be modest, the mechanisms that compensate for these changes may alter the pharmacokinetic and physiological properties of the receptor. It is therefore crucial to understand the subunit composition of individual GABA_A receptors in the diseased brain when developing therapeutics that act at these receptors.     

Prevalence between different alpha subunits performing the benzodiazepine binding sites in native heterologous GABA(A) receptors containing the alpha2 subunit

The presence of two heterologous alpha subunits and a single benzodiazepine binding site in the GABA(A) receptor implicates the existence of pharmacologically active and inactive alpha subunits. This fact raises the question of whether a particular alpha subtype could predominate performing the benzodiazepine binding site. The hippocampal formation expresses high levels of alpha subunits with different benzodiazepine binding properties (alpha1, alpha2 and alpha5). Thus, we first demonstrated the existence of alpha2-alpha1 (36.3 +/- 5.2% of the alpha2 population) and alpha2-alpha5 (20.2 +/- 2.1%) heterologous receptors. A similar alpha2-alpha1 association was observed in cortex. This association allows the direct comparison of the pharmacological properties of heterologous native GABA(A) receptors containing a common (alpha2) and a different (alpha1 or alpha5) alpha subunit. The alpha2 subunit pharmacologically prevailed over the alpha1 subunit in both cortex and hippocampus (there was an absence of high-affinity binding sites for Cl218,872, zolpidem and [3H]zolpidem). This prevalence was directly probed by zolpidem displacement experiments in alpha2-alpha1 double immunopurified receptors (K(i) = 295 +/- 56 nM and 200 +/- 8 nM in hippocampus and cortex, respectively). On the contrary, the alpha5 subunit pharmacologically prevailed over the alpha2 subunit (low- and high-affinity binding sites for zolpidem and [3H]L-655,708, respectively). This prevalence was probed in alpha2-alpha5 double immunopurified receptors. Zolpidem displayed a single low-affinity binding site (K(i) = 1.73 +/- 0.54 microM). These results demonstrated the existence of a differential dominance between the different alpha subunits performing the benzodiazepine binding sites in the native GABA(A) receptors.     

Relative positioning of diazepam in the benzodiazepine-binding-pocket of GABAA receptors

GABA_A receptors are the major inhibitory neurotransmitter receptors in the brain. Some of them are targets of benzodiazepines that are widely used in clinical practice for their sedative/hypnotic, anxiolytic, muscle relaxant and anticonvulsant effects. In order to rationally separate these different drug actions, we need to understand the interaction of such compounds with the benzodiazepine-binding pocket. With this aim, we mutated residues located in the benzodiazepine-binding site individually to cysteine. These mutated receptors were combined with benzodiazepine site ligands carrying a cysteine reactive group in a defined position. Proximal apposition of reaction partners will lead to a covalent reaction. We describe here such proximity-accelerated chemical coupling reactions of α₁S205C and α₁T206C with a diazepam derivative modified at the C-3 position with a reactive isothiocyanate group (–NCS). We also provide new data that identify α₁H101C and α₁N102C as exclusive sites of the reaction of a diazepam derivative where the –Cl atom is replaced by a –NCS group. Based on these observations we propose a relative positioning of diazepam within the benzodiazepine-binding site of α₁β₂γ₂ receptors.     

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.     

Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs

Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2). This comparison was extended to alpha(6)R100Cbeta(2)gamma(2) receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of alpha(6)R100Cbeta(2)gamma(2) enables best interaction with NCS compound, followed by alpha(3)H126Cbeta(2)gamma(2), alpha(1)H101Cbeta(2)gamma(2) and alpha(2)H101Cbeta(2)gamma(2), while alpha(5)H105Cbeta(2)gamma(2) receptors show little interaction. Our results allow conclusions about the relative apposition of alpha(1)H101 and homologous positions in alpha(2), alpha(3), alpha(5) and alpha(6) with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABA(A) receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the alpha/beta subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the alpha/gamma interface.     

Zn2+对爪蟾卵母细胞表达鲫鱼脑GABA受体的调制作用

爪蟾卵母细胞注射鲫鱼脑ｍＲＮＡ后表达的ＧＡＢＡ受体中约８５％为ＧＡＢＡＡ受体。约１５％的成分为ＧＡＮＡＣ受体。本文利用双电极电压箝方法结合药物灌流研究了Ｚｎ６２＋对这两型受体的作用。我们观察到了Ｚｎ＾２＋对它们的调制都是可抑制性的,可逆的。     

Subunit specificity and interaction domain between GABA(A) receptor-associated protein (GABARAP) and GABA(A) receptors

GABARAP (GABA(A) receptor-associated protein) interacts with both microtubules and GABA(A) receptors in vitro and in vivo and is capable of modulating receptor channel kinetics. In this study, we use the intracellular loop of 15 GABA(A) receptor subunits to show that the interaction between GABARAP and GABA(A) receptor is specific for the gamma subunits. Pharmacological characterization of proteins purified by GABARAP affinity column indicates that native GABA(A) receptors interact with GABARAP. Quantitative yeast two-hybrid assays were used to identify the interaction domain in the gamma2 subunit for GABARAP binding, and to identify the interaction domain in GABARAP for GABA(A) receptor binding. A peptide corresponding to the GABARAP interaction domain in the gamma2 subunit was used to inhibit the interaction between GABARAP and the gamma2 subunit. In addition, the ability of GABARAP to promote cluster formation of recombinant receptors expressed in QT-6 fibroblasts was inhibited by a membrane-permeable form of this peptide in a time-dependent manner. The establishment of a model for GABARAP-induced clustering of GABA(A) receptors in living cells and the identification of subunit specificity and interaction domains in the interaction between GABARAP and GABA(A) receptors is a step in dissecting the function of GABARAP in GABA(A) receptor clustering and/or targeting.     

Multiple tyrosine residues at the GABA binding pocket influence surface expression and mediate kinetics of the GABAA receptor

The prevalence of aromatic residues in the ligand binding site of the GABA_A receptor, as with other cys‐loop ligand‐gated ion channels, is undoubtedly important for the ability of neurotransmitters to bind and trigger channel opening. Here, we have examined three conserved tyrosine residues at the GABA binding pocket (β₂Tyr97, β₂Tyr157, and β₂Tyr205), making mutations to alanine and phenylalanine. We fully characterized the effects each mutation had on receptor function using heterologous expression in HEK‐293 cells, which included examining surface expression, kinetics of macroscopic currents, microscopic binding and unbinding rates for an antagonist, and microscopic binding rates for an agonist. The assembly or trafficking of GABA_A receptors was disrupted when tyrosine mutants were expressed as αβ receptors, but interestingly not when expressed as αβγ receptors. Mutation of each tyrosine accelerated deactivation and slowed GABA binding. This provides strong evidence that these residues influence the binding of GABA. Qualitatively, mutation of each tyrosine has a very similar effect on receptor function; however, mutations at β₂Tyr157 and β₂Tyr205 are more detrimental than β₂Tyr97 mutations, particularly to the GABA binding rate. Overall, the results suggest that interactions involving multiple tyrosine residues are likely during the binding process.     

Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABA(A) receptors

Based on a pharmacophore model of the benzodiazepine-binding site of GABA_A receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity ligands of the binding site. For several compounds, K_i values of around 0.20 nM were determined. They show a structural resemblance with the previously described 2-phenyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones (II) and 2-phenyl-[1,2,4]triazolo[1,5-a]quinoxalin-4(5H)-one (III). The 9-bromo substituted compounds 8a-d were prepared in an 8-step synthesis in an overall yield of approximately 40%, and a library of 9-substituted analogues was prepared by cross-coupling reactions. Compound 8e, 21, 22, and 24 were tested on recombinant rat ??₁??₃??₂, ??₂??₃??₂, ??₃??₃??₂, and ??₅??₃??₂ subtypes, and displayed selectivity for the ??₁??₃??₂ isoform.     

Expression of tachykinin receptors in Xenopus oocytes injected with poly(A)~+RNA from cat dorsal root ganglion

ＴａｃｈｙｋｉｎｉｎｆａｍｉｌｙｉｓａｇｒｏｕｐｏｆｎｅｕｒｏｐｅｐｔｉｄｅｓｗｉｔｈｓｉｍｉｌａｒＣｔｅｒｍｉｎａｌｓｅｑｕｅｎｃｅｓａｎｄｒｅｌａｔｅｄｂｉｏａｃｔｉｖｉｔｉｅｓ.ＴｈｅｍａｊｏｒｔａｃｈｙｋｉｎｉｎｓｉｎｍａｍｍａｌｉａｎａｒｅｓｕｂｓｔａｎｃｅＰ(ＳＰ),ｎｅｕｒｏｋｉｎｉｎＡ(ＮＫＡ)ａｎｄｎｅｕｒｏｋｉｎｉｎＢ(ＮＫＢ).Ｃｏｒｒｅｓｐｏｎｄｉｎｇｔｏｔｈｅｓｅｐｅｐｔｉｄｅｓ,ｔｈｒｅｅｄｉｓｔｉｎｃｔｔａｃｈｙｋｉｎｉｎｒｅｃｅｐｔｏｒｓｗｅｒｅｄｉｓｃｏｖｅｒｅｄａｎｄｎ…     

The lack of CB1 receptors prevents neuroadapatations of both NMDA and GABA(A) receptors after chronic ethanol exposure

As the contribution of cannabinoid (CB1) receptors in the neuroadaptations following chronic alcohol exposure is unknown, we investigated the neuroadaptations induced by chronic alcohol exposure on both NMDA and GABA(A) receptors in CB1-/- mice. Our results show that basal levels of hippocampal [(3)H]MK-801 ((1)-5-methyl-10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine) binding sites were decreased in CB1-/- mice and that these mice were also less sensitive to the locomotor effects of MK-801. Basal level of both hippocampal and cerebellar [(3)H]muscimol binding was lower and sensitivity to the hypothermic effects of diazepam and pentobarbital was increased in CB1-/- mice. GABA(A)alpha1, beta2, and gamma2 and NMDA receptor (NR) 1 and 2B subunit mRNA levels were altered in striatum of CB1-/- mice. Our results also showed that [(3)H]MK-801 binding sites were increased in cerebral cortex and hippocampus after chronic ethanol ingestion only in wild-type mice. Chronic ethanol ingestion did not modify the sensitivity to the locomotor effects of MK-801 in both genotypes. Similarly, chronic ethanol ingestion reduced the number of [(3)H]muscimol binding sites in cerebral cortex, but not in cerebellum, only in CB1+/+ mice. We conclude that lifelong deletion of CB1 receptors impairs neuroadaptations of both NMDA and GABA(A) receptors after chronic ethanol exposure and that the endocannabinoid/CB1 receptor system is involved in alcohol dependence.     

Allosteric modulation by single enantiomers of a C3-chiral 1,4-benzodiazepine of the gamma aminobutyric acid type A receptor channel expressed in Xenopus oocytes

Xenopus laevis oocytes injected with Poly(A)⁺-RNA isolated from neuronal tissue express membrane proteins peculiar to the origin of mRNA. The translation of gamma aminobutyric acid type A (GABA_A) receptors has been shown by dose/ response behavior of GABA and the reversible blockade of the GABA-induced current by picrotoxin. This current was analyzed quantitatively under two electrode voltage-clamp conditions. This methodology has been applied for the first time to study the functional properties of the receptor as a function of the stereochemistry of the ligands. The (+)-S and (-)-R enantiomers of a water-soluble benzodiazepine derivative, 7-chloro-1,3-dihydro-3-hemisuccinyloxy-5-phenyl-1,4-benzodiazepin-2-one (OXHEM), obtained by preparative high performance liquid chromatographic (HPLC) resolution on chiral stationary phase, act as agonists in the in vitro modulation of the chloride channel. The (+)-S-OXHEM enantiomer was the more active. Chirality 9:286–290, 1997. © 1997 Wiley-Liss, Inc.     

An interaction between benzodiazepines and neuroactive steroids at GABA A receptors in cultured hippocampal neurons

Neurosteroids are modulators of several receptors and ion channels and are implicated in the pathophysiology of several neuropsychiatric diseases including hepatic encephalopathy (HE). The neurosteroid, allopregnanolone, a positive allosteric modulator of GABA_A receptors, accumulates in the brains of HE patients where it can potentiate GABA_A receptor-mediated responses. Attenuation of the effects of neurosteroids on GABA-ergic neurotransmission is therefore of interest for the management of HE. In the present study, we determined the effect of the benzodiazepine partial inverse agonist, Ro15-4513, and the benzodiazepine antagonist, flumazenil on modulation of the GABA_A mediated chloride currents by allopregnanolone and on spontaneous synaptic activity in cultured hippocampal neurons using the patch-clamp technique. Allopregnanolone (0.03–0.3 μM), dose-dependently potentiated GABA-induced currents, an action significantly reduced by Ro15-4513 (10 μM). In contrast, flumazenil (10 μM) had no effect on the ability of allopregnanolone to potentiate GABA_A currents but it blocked the effects of Ro15-4513. The frequency of spontaneous synaptic activity was significantly reduced in the presence of allopregnanolone (0.1 μM) from 1.5 ± 0.7 to 0.1 ± 0.04 Hz. This action was partially reversed by Ro15-4513 (10 μM) but was not significantly influenced by flumazenil (10 μM). These findings suggest that the beneficial affects of Ro15-4513 in experimental HE result from attenuation of the effects of neurosteroids at GABA_A receptors. Our results may provide a rational basis for the use of benzodiazepine inverse agonists in the management and treatment of hepatic encephalopathy in patients with liver failure.     

(+)- and (-)-cis-2-aminomethylcyclopropanecarboxylic acids show opposite pharmacology at recombinant rho(1) and rho(2) GABA(C) receptors

The effects of the enantiomers of (+/-)-CAMP and (+/-)-TAMP [(+/-)-cis- and (+/-)-trans-2-aminomethylcyclopropanecarboxylic acids, respectively], which are cyclopropane analogues of GABA, were tested on GABA(A) and GABA(C) receptors expressed in Xenopus laevis oocytes using two-electrode voltage clamp methods. (+)-CAMP was found to be a potent and full agonist at homooligomeric GABA(C) receptors (K:(D) approximately 40 microM: and I:(max) approximately 100% at rho(1); K:(D) approximately 17 microM: and I:(max) approximately 100% at rho(2)) but a very weak antagonist at alpha(1)beta(2)gamma(2L) GABA(A) receptors. In contrast, (-)-CAMP was a very weak antagonist at both alpha(1)beta(2)gamma(2L) GABA(A) receptors and homooligomeric GABA(C) receptors (IC(50) approximately 900 microM: at rho(1) and approximately 400 microM: at rho(2)). Furthermore, (+)-CAMP appears to be a superior agonist to the widely used GABA(C) receptor partial agonist cis-4-aminocrotonic acid (K:(D) approximately 74 microM: and I:(max) approximately 78% at rho(1); K:(D) approximately 70 microM: and I:(max) approximately 82% at rho(2)). (-)-TAMP was the most potent of the cyclopropane analogues on GABA(C) receptors (K:(D) approximately 9 microM: and I:(max) approximately 40% at rho(1); K:(D) approximately 3 microM: and I:(max) approximately 50-60% at rho(2)), but it was also a moderately potent GABA(A) receptor partial agonist (K:(D) approximately 50-60 microM: and I:(max) approximately 50% at alpha(1)beta(2)gamma(2L) GABA(A) receptors). (+)-TAMP was a less potent partial agonist at GABA(C) receptors (K:(D) approximately 60 microM: and I:(max) approximately 40% at rho(1); K:(D) approximately 30 microM: and I:(max) approximately 60% at rho(2)) and a weak partial agonist at alpha(1)beta(2)gamma(2L) GABA(A) receptors (K:(D) approximately 500 micro: and I:(max) approximately 50%). None of the isomers of (+/-)-CAMP and (+/-)-TAMP displayed any interaction with GABA transport at the concentrations tested. Molecular modeling based on the present results provided new insights into the chiral preferences for either agonism or antagonism at GABA(C) receptors.     

An intracellular motif of P2X(3) receptors is required for functional cross-talk with GABA(A) receptors in nociceptive DRG neurons

Functional cross-talk between structurally unrelated P2X ATP receptors and members of the 'cys-loop' receptor-channel superfamily represents a recently-discovered mechanism for rapid modulation of information processing. The extent and the mechanism of the inhibitory cross-talks between these two classes of ionotropic receptors remain poorly understood, however. Both ionic and molecular coupling were proposed to explain cross-inhibition between P2X subtypes and GABA(A) receptors, suggesting a P2X subunit-dependent mechanism. We show here that cross-inhibition between neuronal P2X(3) or P2X(2+3) and GABA(A) receptors does not depend on chloride and calcium ions. We identified an intracellular QST(386-388) motif in P2X(3) subunits which is required for the functional coupling with GABA(A) receptors. Moreover the cross-inhibition between native P2X(3) and GABA receptors in cultured rat dorsal root ganglia (DRG) neurons is abolished by infusion of a peptide containing the QST motif as well as by viral expression of the main intracellular loop of GABA(A)beta3 subunits. We provide evidence that P2X(3) and GABA(A) receptors are colocalized in the soma and central processes of nociceptive DRG neurons, suggesting that specific intracellular P2X(3)-GABA(A) subunit interactions underlie a pre-synaptic cross-talk that might contribute to the regulation of sensory synaptic transmission in the spinal cord.     

Chronic benzodiazepine treatment of cells expressing recombinant GABAA receptors uncouples allosteric binding: studies on possible mechanisms

Functional and behavioral tolerance to chronic benzodiazepine (BZ) exposure has been associated with an uncoupling of the BZ and GABA binding sites. As in rats exposed to BZ for periods of a week or longer, recombinant GABA(A) receptors (GABARs) expressed in Sf9 cells lose the normally observed allosteric enhancement of [3H]flunitrazepam binding by GABA agonists, which is measured in homogenized membranes after a few hours exposure to pharmacological doses of agonist BZ. Treatment of Sf9 cells expressing recombinant GABAR with various drugs that inhibit protein kinase A (PKA), but not protein kinase C (PKC), resulted in an uncoupling of the BZ and GABA binding sites; whereas promotion of phosphorylation by PKA, but not PKC, favored coupling and recoupling. However, mutation of the only PKA phosphorylation site expressed from among the subunits proved that direct phosphorylation of the GABAR was not involved in either coupling after chronic BZ exposure or reversal of uncoupling after exposure to the competitive BZ antagonist, flumazenil. Osmotic-shock of cell membrane homogenates to lyse intracellular compartments reversed uncoupling, and uncoupling can be replicated in untreated cells by performing membrane binding assays in an acidic environment, suggesting that GABARs become internalized into an acidic intracellular environment where normal BZ binding occurs, but that potentiation by GABA is hindered. The internalization of receptors was shown by immunofluorescence after chronic exposure to either BZ or the PKA inhibitor H-89.     

A method for the determination of the integrity of labeled GABA used in membrane receptor binding assay

A simple method for the determination of the proportion of true GABA within labeled GABA used for membrane binding assay is presented. The method is intended for the assessment of the integrity of refrigerator (+4°C) stored labeled neurotransmitter. Its application allows a precise determination of the binding parameters.