Search

The first two models of the Sea and Land Surface Temperature Radiometers (SLSTR) for the European Copernicus Sentinel-3 missions were tested prior to launch at the Rutherford Appleton Laboratory space instrument calibration facility. The pre-launch tests provide an essential reference that ensures that the flight data of SLSTR are calibrated to the same standards needed for surface temperature measurements and to those used by shipborne radiometers for Fiducial Reference Measurement (FRM). The radiometric calibrations of the thermal infrared channels were validated against accurate and traceable reference BB sources under flight representative thermal vacuum environment. Measurements were performed in both earth views for source temperatures covering the main operating range, for different instrument configurations and for the full field-of-view of the instruments. The data were used to derive non-linearity curves to be used in the level-1 processing. All results showed that the measured brightness temperatures and radiometric noise agreed within the requirements for the mission. An inconsistency that particularly affected SLSTR-A was observed which has been attributed to an internal stray light error. A correction for the stray light has been proposed to reduce the error. The internal stray light error was reduced for SLSTR-B by replacing the coating on the main aperture stop. We present a description of the test methodology and the key results.

Providing uncertainties in satellite datasets used for Earth observation can be a daunting prospect because of the many processing stages and input data required to convert raw detector counts to calibrated radiances. The Sea and Land Surface Temperature Radiometer (SLSTR) was designed to provide measurements of the Earth’s surface for operational and climate applications. In this paper the authors describe the traceability chain and derivation of uncertainty estimates for the thermal infrared channel radiometry. Starting from the instrument model, the contributing input quantities are identified to build up an uncertainty effects tree. The characterisation of each input effect is described, and uncertainty estimates provided which are used to derive the combined uncertainties as a function of scene temperature. The SLSTR Level-1 data products provide uncertainty estimates for fully random effects (noise) and systematic effects that can be mapped for each image pixel, examples of which are shown.

The &alpha, 7 nicotinic acetylcholine receptor (&alpha, 7 nAChR) is involved in several cognitive and physiologic processes, its expression levels and patterns change in neurologic and psychiatric diseases, such as schizophrenia and Alzheimer&rsquo, s disease, which makes it a relevant drug target. Development of selective radioligands is important for defining binding properties and occupancy of novel molecules targeting the receptor. We tested the in vitro binding properties of [125I]Iodo-ASEM [(3-(1,4-diazabycyclo[3.2.2]nonan-4-yl)-6-(125I-iododibenzo[b,d]thiopentene 5,5-dioxide)] in the mouse, rat and pig brain using autoradiography. The in vivo binding properties of [18F]ASEM were investigated using positron emission tomography (PET) in the pig brain. [125I]Iodo-ASEM showed specific and displaceable high affinity (~1 nM) binding in mouse, rat, and pig brain. Binding pattern overlapped with [125I]&alpha, bungarotoxin, specific binding was absent in &alpha, 7 nAChR gene-deficient mice and binding was blocked by a range of &alpha, 7 nAChR orthosteric modulators in an affinity-dependent order in the pig brain. Interestingly, relative to the wild-type, binding in &beta, 2 nAChR gene-deficient mice was lower for [125I]Iodo-ASEM (58% &plusmn, 2.7%) than [125I]&alpha, bungarotoxin (23% &plusmn, 0.2%), potentially indicating different binding properties to heteromeric &alpha, 7&beta, 2 nAChR. [18F]ASEM PET in the pig showed high brain uptake and reversible tracer kinetics with a similar spatial distribution as previously reported for &alpha, 7 nAChR. Blocking with SSR-180,711 resulted in a significant decrease in [18F]ASEM binding. Our findings indicate that [125I]Iodo-ASEM allows sensitive and selective imaging of &alpha, 7 nAChR in vitro, with better signal-to-noise ratio than previous tracers. Preliminary data of [18F]ASEM in the pig brain demonstrated principal suitable kinetic properties for in vivo quantification of &alpha, 7 nAChR, comparable to previously published data.

By structure–activity relationship studies on the tilorone scaffold, the ‘one armed’ substituted dibenzothiophenes and the fluoren-9-ones were identified as the most potential α7 nAChR ligands. While the suitability of dibenzothiophene derivatives as PET tracers is recognized, the potential of fluoren-9-ones is insufficiently investigated. We herein report on a series of fluoren-9-one based derivatives targeting α7 nAChR with compounds 8a and 8c possessing the highest affinity and selectivity. Accordingly, with [18F]8a and [18F]8c we designed and initially evaluated the first fluoren-9-one derived α7 nAChR selective PET ligands. A future application of these radioligands is facilitated by the herein presented successful implementation of fully automated radiosynthesis.

Changes in the expression of α7 nicotinic acetylcholine receptors (α7 nAChRs) in the human brain are widely assumed to be associated with neurological and neurooncological processes. Investigation of these receptors in vivo depends on the availability of imaging agents such as radioactively labelled ligands applicable in positron emission tomography (PET). We report on a series of new ligands for α7 nAChRs designed by the combination of dibenzothiophene dioxide as a novel hydrogen bond acceptor functionality with diazabicyclononane as an established cationic center. To assess the structure-activity relationship (SAR) of this new basic structure, we further modified the cationic center systematically by introduction of three different piperazine-based scaffolds. Based on in vitro binding affinity and selectivity, assessed by radioligand displacement studies at different rat and human nAChR subtypes and at the structurally related human 5-HT3 receptor, we selected the compound 7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-2-fluorodibenzo-[b,d]thiophene 5,5-dioxide (10a) for radiolabeling and further evaluation in vivo. Radiosynthesis of [18F]10a was optimized and transferred to an automated module. Dynamic PET imaging studies with [18F]10a in piglets and a monkey demonstrated high uptake of radioactivity in the brain, followed by washout and target-region specific accumulation under baseline conditions. Kinetic analysis of [18F]10a in pig was performed using a two-tissue compartment model with arterial-derived input function. Our initial evaluation revealed that the dibenzothiophene-based PET radioligand [18F]10a ([18F]DBT-10) has high potential to provide clinically relevant information about the expression and availability of α7 nAChR in the brain.

Deciphering which specific agonist-receptor interactions affect efficacy levels is of high importance, because this will ultimately aid in designing selective drugs. The novel compound NS3861 and cytisine are agonists of nicotinic acetylcholine receptors (nAChRs) and both bind with high affinity to heteromeric α3β4 and α4β2 nAChRs. However, initial data revealed that the activation patterns of the two compounds show very distinct maximal efficacy readouts at various heteromeric nAChRs. To investigate the molecular determinants behind these observations, we performed in-depth patch clamp electrophysiological measurements of efficacy levels at heteromeric combinations of α3- and α4-, with β2- and β4-subunits, and various chimeric constructs thereof. Compared with cytisine, which selectively activates receptors containing β4- but not β2-subunits, NS3861 displays the opposite β-subunit preference and a complete lack of activation at α4-containing receptors. The maximal efficacy of NS3861 appeared solely dependent on the nature of the ligand-binding domain, whereas efficacy of cytisine was additionally affected by the nature of the β-subunit transmembrane domain. Molecular docking to nAChR subtype homology models suggests agonist specific interactions to two different residues on the complementary subunits as responsible for the β-subunit preference of both compounds. Furthermore, a principal subunit serine to threonine substitution may explain the lack of NS3861 activation at α4-containing receptors. In conclusion, our results are consistent with a hypothesis where agonist interactions with the principal subunit (α) primarily determine binding affinity, whereas interactions with key amino acids at the complementary subunit (β) affect agonist efficacy.

BACKGROUND AND PURPOSE Positive allosteric modulation of α4β2 nicotinic acetylcholine (nACh) receptors could add a new dimension to the pharmacology and therapeutic approach to these receptors. The novel modulator NS9283 was therefore tested extensively.

The α4β2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of α4β2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A correlation between binding affinities at α4β2 and the acetylcholine-binding protein from Lymnaea stagnalis (Ls-AChBP) confirms Ls-AChBP as structural surrogate for α4β2 receptors. Crystal structures of five agonists with efficacies at α4β2 from 21–76% were determined in complex with Ls-AChBP. No variation in closure of loop C is observed despite large efficacy variations. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong intersubunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong intersubunit anchoring.

Background and purpose: Several agonists of the α7 nicotinic acetylcholine receptor (nAChR) have been developed for treatment of cognitive deficits. However, agonist efficacy in vivo is difficult to reconcile with rapid α7 nAChR desensitization in vitro; and furthermore, the correlation between in vitro receptor efficacy and in vivo behavioural efficacy is not well delineated. The possibility that agonists of this receptor actually function in vivo as inhibitors via desensitization has not been finally resolved. Experimental approach: Two structurally related α7 nAChR agonists were characterized and used to assess the degree of efficacy required in a behavioural paradigm. Key results: NS6784 activated human and rat α7 nAChR with EC50s of 0.72 and 0.88 µM, and apparent efficacies of 77 and 97% respectively. NS6740, in contrast, displayed little efficacy at α7 nAChR (

Binding studies initially suggested that the muscarinic agonist, xanomeline, was a subtype selective muscarinic M (1) receptor agonist, and a potential new treatment for Alzheimer's disease. However, later in vitro and in vivo functional studies suggest that this compound is probably better described as a subtype selective M(1)/M(4) muscarinic receptor agonist. This subtype selectivity profile has been claimed to explain the limited classical cholinomimetic side effects, particularly gastrointestinal, seen with xanomeline in animals. However, in both healthy volunteers and Alzheimer's patients many of these side effects have been reported for xanomeline and in the patient population this led to a>50% discontinuation rate. Clearly, the preclinical studies have not been able to predict this adverse profile of xanomeline, and this suggests that either xanomeline is not as subtype selective as predicted from preclinical research or that there are differences between humans and animals with regard to muscarinic receptors. Nevertheless, in Alzheimer's patients xanomeline dose‐dependently improves aspects of behavioral disturbance and social behavior including a reduction in hallucinations, agitation, delusions, vocal outbursts and suspiciousness. The effects on cognition are not as robust and mainly seen at the highest doses tested. These effects in Alzheimer's patients have given impetus to the suggestion that muscarinic agonists have potential antipsychotic effects. The current review assesses the antipsychotic profile of xanomeline within the framework of the limited clinical studies with cholinergic agents in man, and the preclinical research on xanomeline using various models commonly used for the assessment of new antipsychotic drugs. In general, xanomeline has an antipsychotic‐like profile in various dopamine models of psychosis and this agrees with the known interactions between the cholinergic and dopaminergic systems in the brain. Moreover, current data suggests that the actions of xanomeline at the M(4) muscarinic receptor subtype might mediate its antidopaminergic effects. Particularly intriguing are studies showing that xanomeline, even after acute administration, selectively inhibits the firing of mesolimbic dopamine cells relative to dopamine cell bodies projecting to the striatum. This data suggest that xanomeline would have a faster onset of action compared to current antipsychotics and would not induce extrapyramidal side effects. The preclinical data on the whole are promising for an antipsychotic‐like profile. If in a new formulation (i.e., transdermal) xanomeline has less adverse effects, this drug may be valuable in the treatment of patients with psychosis.

The synthesis of unsaturated unnatural amino acids according to the Heck-Jeffery protocol using a ball-milling procedure is presented. NOE data recorded on the products confirm that the Z-isomer is formed. This type of mechano-chemistry provides an efficient, "solvent free" and reliable method for the synthesis of substituted dehydroalanines. These conditions provide patterns of reactivity complementary to conventional procedures. (C) 2004 Elsevier B.V. All rights reserved.

NS 1231 [5-(4-chlorophenyl)-6,7,8,9-tetrahydro-1H-pyrrolo-[3.2-h]naphthalene-2,3-dione-3-oxime] belongs to a chemical series of compounds, which exhibit neurotrophic-like activities. In vitro, NS 1231 rescued nerve growth factor (NGF)-differentiated PC12 cells from death induced by withdrawal of trophic factors. In addition, NS 1231 stimulated NGF-induced neurite outgrowth of undifferentiated PC12 cells. At the molecular level, NS 1231 enhanced NGF-induced signalling events, such as TrkA phosphorylation at the Shc-binding site Tyr490 as well as ERK activation in PC12 cells. Moreover, NS 1231 reduced NMDA-induced excitotoxicity in organotypic hippocampal slice cultures. In a gerbil model of transient global ischaemia, treatment with NS 1231 reduced the delayed loss of neurons in the hippocampal CA1 layer. Furthermore, NS 1231 treatment resulted in a 43% reduction in total infarct volume in the mouse middle cerebral artery occlusion (MCAO) model. The present data thus implicate a therapeutic potential of NS 1231 or structural analogues in treatment of cerebral ischaemia.

Background The α7 nicotinic acetylcholine receptor (nAChR) is an important molecular target in neuropsychiatry and oncology. Development of applicable highly specific radiotracers has been challenging due to comparably low protein expression. To identify novel ligands as candidates for positron emission tomography (PET), a library of diazabicyclononane compounds was screened regarding affinity and specificity towards α7 nAChRs. From these, [18F]NS14490 has been shown to yield reliable results in organ distribution studies; however, the radiosynthesis of [18F]NS14490 required optimization and automation to obtain the radiotracer in quantities allowing dynamic PET studies in piglets. Methods Automated radiosynthesis of [18F]NS14490 has been performed by [18F]fluorination with the tosylate precursor in the TRACERlab™ FX F-N synthesis module (Waukesha, WI, USA). After optimization, the radiochemical yield of [18F]NS14490 was consistently approximately 35%, and the total synthesis time was about 90 min. The radiotracer was prepared with >92% radiochemical purity, and the specific activity at the end of the synthesis was 226 ± 68 GBq μmol−1. PET measurements were performed in young pigs to investigate the metabolic stability and cerebral binding of [18F]NS14490 without and with administration of the α7 nAChR partial agonist NS6740 in baseline and blocking conditions. Results The total distribution volume relative to the metabolite-corrected arterial input was 3.5 to 4.0 mL g−1 throughout the telencephalon and was reduced to 2.6 mL g−1 in animals treated with NS6740. Assuming complete blockade, this displacement indicated a binding potential (BPND) of approximately 0.5 in the brain of living pigs. In addition, evidence for specific binding in major brain arteries has been obtained. Conclusion [18F]NS14490 is not only comparable to other preclinically investigated PET radiotracers for imaging of α7 nAChR in brain but also could be a potential PET radiotracer for imaging of α7 nAChR in vulnerable plaques of diseased vessels.

A convenient method for the synthesis of 10-methyl- 8,10-diazabicyclo[4.3.1]decane, possessing a novel diazabicyclic ring system, as an important synthetic organic chemistry building block was developed using octanedioic acid as a starting material. The key transformation in the 5-step synthesis sequence involved a reaction of dimethyl 2,7-dibromooctanoate with methylamine, which resulted in the formation of cis -dimethyl 1-methylazepan-2,7-dicarboxylate. The lat ter was further transformed into bicyclic 8-benzyl-10-m ethyl-8,10-diazabicyclo[4.3.1]decane-7,9dione under heating with benzylamine. Reduction of the formed bicyclic dione with lithium aluminium hydride resulted in 8-benzyl-10-methyl-8,10-diazabicyclo[4.3.1]decane, and hydrogenolysis efficiently yielded the target produ ct.

The α4β2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of α4β2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A correlation between binding affinities at α4β2 and the acetylcholine-binding protein from Lymnaea stagnalis (Ls-AChBP) confirms Ls-AChBP as structural surrogate for α4β2 receptors. Crystal structures of five agonists with efficacies at α4β2 from 21-76% were determined in complex with Ls-AChBP. No variation in closure of loop C is observed despite large efficacy variations. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong intersubunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong intersubunit anchoring.

Small-molecule α(7) nicotinic acetylcholine receptor (α(7)nAChR) agonists are currently validated for use as treatment for cognitive disturbances in schizophrenia and in Alzheimer disease. A suitable radiolabeled α(7)nAChR PET tracer would be important for in vivo quantification of α(7)nAChR binding in humans and to measure α(7)nAChR occupancy of α(7)nAChR drug candidates. Here, we present the radiosynthesis and in vivo evaluation of (11)C-NS14492 as a selective α(7)nAChR PET radioligand.The high-affinity α(7)nAChR-selective partial agonist NS14492 was radiolabeled by methylation of its desmethyl precursor using (11)C-methyl triflate. Female Danish Landrace pigs were studied at baseline and after intravenous administration of blocking doses of either the α(7)nAChR partial agonist SSR180711 or the unlabeled NS14492. (11)C-NS14492 was given as an intravenous bolus injection, and the pigs were scanned for 90 min both at baseline and in the blocked conditions. Arterial blood was collected during the scanning, plasma was counted, and parent compound fraction was determined with radio-high-performance liquid chromatography. PET data were quantified with a graphical analysis with arterial input; (11)C-NS14492 regional distribution volumes were calculated, and α(7)nAChR occupancy was determined using an occupancy plot.(11)C-NS14492 had a high uptake in the pig brain, with the highest binding in the cerebral cortex and thalamus in accordance with α(7)nAChR distribution. Pretreatment with NS14492 and SSR180711 consistently decreased distribution volumes of (11)C-NS14492 in all examined regions, in a dose-dependent manner, supporting the finding that the radioligand binds selectively to α(7)nAChR in vivo.We report here that (11)C-NS14492 is the first, to our knowledge, PET radioligand for α(7)nAChR showing a dose-dependent decline in cerebral binding after receptor blockade. This compound is considered a promising PET tracer for in vivo measurements of α(7)nAChR binding in the human brain.

The neuronal α4β2 nicotinic acetylcholine receptors exist as two distinct subtypes, (α4)(2)(β2)(3) and (α4)(3)(β2)(2), and biphasic responses to acetylcholine and other agonists have been ascribed previously to coexistence of these two receptor subtypes. We offer a novel and radical explanation for the observation of two distinct agonist sensitivities. Using different expression ratios of mammalian α4 and β2 subunits and concatenated constructs, we demonstrate that a biphasic response is an intrinsic functional property of the (α4)(3)(β2)(2) receptor. In addition to two high-sensitivity sites at α4β2 interfaces, the (α4)(3)(β2)(2) receptor contains a third low-sensitivity agonist binding site in the α4α4 interface. Occupation of this site is required for full activation and is responsible for the widened dynamic response range of this receptor subtype. By site-directed mutagenesis, we show that three residues, which differ between the α4β2 and α4α4 sites, control agonist sensitivity. The results presented here provide a basic insight into the function of pentameric ligand-gated ion channels, which enables modulation of the receptors with hitherto unseen precision; it becomes possible to rationally design therapeutics targeting subpopulations of specific receptor subtypes.

Proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) domains play key roles in the assembly and regulation of cellular signaling pathways and represent putative targets for new pharmacotherapeutics. Here we describe the first small-molecule inhibitor (FSC231) of the PDZ domain in protein interacting with C kinase 1 (PICK1) identified by a screening of ~44,000 compounds in a fluorescent polarization assay. The inhibitor bound the PICK1 PDZ domain with an affinity similar to that observed for endogenous peptide ligands ( K i ~10.1 μM). Mutational analysis, together with computational docking of the compound in simulations starting from the PDZ domain structure, identified the binding mode of FSC231. The specificity of FSC231 for the PICK1 PDZ domain was supported by the lack of binding to PDZ domains of postsynaptic density protein 95 (PSD-95) and glutamate receptor interacting protein 1 (GRIP1). Pretreatment of cultured hippocampal neurons with FSC231 inhibited coimmunopreciptation of the AMPA receptor GluR2 subunit with PICK1. In agreement with inhibiting the role of PICK1 in GluR2 trafficking, FSC231 accelerated recycling of pHluorin-tagged GluR2 in hippocampal neurons after internalization in response to NMDA receptor activation. FSC231 blocked the expression of both long-term depression and long-term potentiation in hippocampal CA1 neurons from acute slices, consistent with inhibition of the bidirectional function of PICK1 in synaptic plasticity. Given the proposed role of the PICK1/AMPA receptor interaction in neuropathic pain, excitotoxicity, and cocaine addiction, FSC231 might serve as a lead in the future development of new therapeutics against these conditions.

Several agonists of the alpha7 nicotinic acetylcholine receptor (nAChR) have been developed for treatment of cognitive deficits. However, agonist efficacy in vivo is difficult to reconcile with rapid alpha7 nAChR desensitization in vitro; and furthermore, the correlation between in vitro receptor efficacy and in vivo behavioural efficacy is not well delineated. The possibility that agonists of this receptor actually function in vivo as inhibitors via desensitization has not been finally resolved.Two structurally related alpha7 nAChR agonists were characterized and used to assess the degree of efficacy required in a behavioural paradigm.NS6784 activated human and rat alpha7 nAChR with EC(50)s of 0.72 and 0.88 microM, and apparent efficacies of 77 and 97% respectively. NS6740, in contrast, displayed little efficacy at alpha7 nAChR (2% in oocytes,or =8% in GH4C1 cells), although its agonist-like properties were revealed by adding a positive allosteric modulator of alpha7 nAChRs or using the slowly desensitizing alpha7V274T receptor. In mouse inhibitory avoidance (IA) memory retention, NS6784 enhanced performance as did the 60% partial agonist A-582941. In contrast, NS6740 did not enhance performance, but blocked effects of A-582941.Collectively, these findings suggest that a degree of alpha7 nAChR agonist efficacy is required for behavioural effects in the IA paradigm, and that such behavioural efficacy is not due to alpha7 nAChR desensitization. Also, a partial agonist of very low efficacy for this receptor could be used as an inhibitor, in the absence of alpha7 nAChR antagonists with favourable CNS penetration.

The release of mitochondrial proapoptotic proteins into the cytosol is the key event in apoptosis signaling, leading to the activation of caspases. Once in the cytosol, cytochrome c triggers the formation of a caspase-activating protein complex called the apoptosome, whereas Smac/Diablo and Omi/htra2 antagonize the caspase inhibitory effect of inhibitor of apoptosis proteins (IAPs). Here, we identify diarylurea compounds as effective inhibitors of the cytochrome c-induced formation of the active, approximately 700-kDa apoptosome complex and caspase activation. Using diarylureas to inhibit the formation of the apoptosome complex, we demonstrated that cytochrome c, rather than IAP antagonists, is the major mitochondrial caspase activation factor in tumor cells treated with tumor necrosis factor. Thus, we have identified a novel class of compounds that inhibits apoptosis by blocking the activation of the initiator caspase 9 by directly inhibiting the formation of the apoptosome complex. This mechanism of action is different from that employed by the widely used tetrapeptide inhibitors of caspases or known endogenous apoptosis inhibitors, such as Bcl-2 and IAPs. Thus, these compounds provide a novel specific tool to investigate the role of the apoptosome in mitochondrion-dependent death paradigms.

Positive allosteric modulators (PAMs) of α4β2 nicotinic acetylcholine receptors have the potential to improve cognitive function and alleviate pain. However, only a few selective PAMs of α4β2 receptors have been described limiting both pharmacological understanding and drug-discovery efforts. Here, we describe a novel selective PAM of α4β2 receptors, NS206, and compare with a previously reported PAM, NS9283. Using two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, NS206 was observed to positively modulate acetylcholine (ACh)-evoked currents at both known α4β2 stoichiometries (2α:3β and 3α:2β). In the presence of NS206, peak current amplitudes surpassed those of maximal efficacious ACh stimulations (Emax(ACh)) with no or limited effects at potencies and current waveforms (as inspected visually). This pharmacological action contrasted with that of NS9283, which only modulated the 3α:2β receptor and acted by left shifting the ACh concentration-response relationship. Interestingly, the two modulators can act simultaneously in an additive manner at 3α:2β receptors, which results in current levels exceeding Emax(ACh) and a left-shifted ACh concentration-response relationship. Through use of chimeric and point-mutated receptors, the binding site of NS206 was linked to the α4-subunit transmembrane domain, whereas binding of NS9283 was shown to be associated with the αα-interface in 3α:2β receptors. Collectively, these data demonstrate the existence of two distinct modulatory sites in α4β2 receptors with unique pharmacological attributes that can act additively. Several allosteric sites have been identified within the family of Cys-loop receptors and with the present data, a detailed picture of allosteric modulatory mechanisms of these important receptors is emerging.