Rat Α7 Research Articles

Pharmacological and behavioral profile of N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-6-chinolincarboxamide (EVP-5141), a novel α7 nicotinic acetylcholine receptor agonist/serotonin 5-HT3 receptor antagonist

Agonists of α7 nicotinic acetylcholine receptors (nAChRs) may have therapeutic potential for the treatment of cognitive deficits. This study describes the in vitro pharmacology of the novel α7 nAChR agonist/serotonin 5-HT3 receptor (5-HT3R) antagonist N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-6-chinolincarboxamide (EVP-5141) and its behavioral effects. EVP-5141 bound to α7 nAChRs in rat brain membranes (K i = 270nM) and to recombinant human serotonin 5-HT3Rs (K i = 880nM) but had low affinity for α4β2 nAChRs (K i > 100μM). EVP-5141 was a potent agonist at recombinant rat and human α7 nAChRs expressed in Xenopus oocytes. EVP-5141 acted as 5-HT3R antagonist but did not block α3β4, α4β2, and muscle nAChRs. Rats trained to discriminate nicotine from vehicle did not generalize to EVP-5141 (0.3-30mg kg(-1), p.o.), suggesting that the nicotine cue is not mediated by the α7 nAChR and that EVP-5141 may not share the abuse liability of nicotine. EVP-5141 (0.3-3mg kg(-1)) improved performance in the rat social recognition test. EVP-5141 (0.3mg kg(-1), p.o.) ameliorated scopolamine-induced retention deficits in the passive avoidance task in rats. EVP-5141 (1mg kg(-1), i.p.) improved spatial working memory of aged (26- to 32-month-old) rats in a water maze repeated acquisition task. In addition, EVP-5141 improved both object and social recognition memory in mice (0.3mg kg(-1), p.o.). EVP-5141 improved performance in several learning and memory tests in both rats and mice, supporting the hypothesis that α7 nAChR agonists may provide a novel therapeutic strategy for the treatment of cognitive deficits in Alzheimer's disease or schizophrenia.

Design, Synthesis, and Pharmacological Characterization of Novel Spirocyclic Quinuclidinyl‐Δ2‐Isoxazoline Derivatives as Potent and Selective Agonists of α7 Nicotinic Acetylcholine Receptors

A set of racemic spirocyclic quinuclidinyl-Δ(2)-isoxazoline derivatives was synthesized using a 1,3-dipolar cycloaddition-based approach. Target compounds were assayed for binding affinity toward rat neuronal homomeric (α7) and heteromeric (α4β2) nicotinic acetylcholine receptors. Δ(2) -Isoxazolines 3 a (3-Br), 6 a (3-OMe), 5 a (3-Ph), 8 a (3-OnPr), and 4 a (3-Me) were the ligands with the highest affinity for the α7 subtype (K(i) values equal to 13.5, 14.2, 25.0, 71.6, and 96.2 nM, respectively), and showed excellent α7 versus α4β2 subtype selectivity. These compounds, tested in electrophysiological experiments against human α7 and α4β2 receptors stably expressed in cell lines, behaved as partial α7 agonists with varying levels of potency. The two enantiomers of (±)-3-methoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene sesquifumarate 6 a were prepared using (+)-dibenzoyl-L- or (-)-dibenzoyl-D-tartaric acid as resolving agents. Enantiomer (R)-(-)-6 a was found to be the eutomer, with K(i) values of 4.6 and 48.7 nM against rat and human α7 receptors, respectively.

Identifying the Binding Site of Novel Methyllycaconitine (MLA) Analogs at α4β2 Nicotinic Acetylcholine Receptors

Neuronal nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels that mediate fast synaptic transmission. Methyllycaconitine (MLA) is a selective and potent antagonist of the α7 nAChR, and its anthranilate ester side-chain is important for its activity. Here we report the influence of structure on nAChR inhibition for a series of novel MLA analogs, incorporating either an alcohol or anthranilate ester side-chain to an azabicyclic or azatricyclic core against rat α7, α4β2, and α3β4 nAChRs expressed in Xenopus oocytes. The analogs inhibited ACh (EC(50)) within an IC(50) range of 2.3-26.6 μM. Most displayed noncompetitive antagonism, but the anthranilate ester analogs exerted competitive behavior at the α7 nAChR. At α4β2 nAChRs, inhibition by the azabicyclic alcohol was voltage-dependent suggesting channel block. The channel-lining residues of α4 subunits were mutated to cysteine and the effect of azabicyclic alcohol was evaluated by competition with methanethiosulfonate ethylammonium (MTSEA) and a thiol-reactive probe in the open, closed, and desensitized states of α4β2 nAChRs. The azabicyclic alcohol was found to compete with MTSEA between residues 6' and 13' in a state-dependent manner, but the reactive probe only bonded with 13' in the open state. The data suggest that the 13' position is the dominant binding site. Ligand docking of the azabicyclic alcohol into a (α4)(3)(β2)(2) homology model of the closed channel showed that the ligand can be accommodated at this location. Thus our data reveal distinct pharmacological differences between different nAChR subtypes and also identify a specific binding site for a noncompetitive channel blocker.

Chemical synthesis and characterization of two α4/7-conotoxins

α-Conotoxins are small disulfide-constrained peptides that act as potent and selective antagonists on specific subtypes of nicotinic acetylcholine receptors (nAChRs). We previously cloned two α-conotoxins, Mr1.1 from the molluscivorous Conus marmoreus and Lp1.4 from the vermivorous Conus leopardus. Both of them have the typical 4/7-type framework of the subfamily of α-conotoxins that act on neuronal nAChRs. In this work, we chemically synthesized these two toxins and characterized their functional properties. The synthetic Mr1.1 could primarily inhibit acetylcholine (ACh)-evoked currents reversibly in the oocyte-expressed rat α7 nAChR, whereas Lp1.4 was an unexpected specific blocker of the mouse fetal muscle α1β1γδ receptor. Although their inhibition affinities were relatively low, their unique receptor recognition profiles make them valuable tools for toxin-receptor interaction studies. Mr1.1 could also suppress the inflammatory response to pain in vivo, suggesting that it should be further investigated with respect to its molecular role in analgesia and its mechanism or therapeutic target for the treatment of pain.

In vitro binding characteristics of [ 3H]AZ11637326, a novel α7-selective neuronal nicotinic receptor agonist radioligand

AZ11637326 (5′-(2-fluoro[3,4,5 −3H3]phenyl)-spiro[1-azabicyclo [2.2.2]octane-3,2′(3′H)-furo[2,3-b]pyridine) is a potent partial agonist at the human α7 neuronal nicotinic receptor with sub-nanomolar affinity for the human and rat α7 [ 125I]α-bungarotoxin binding sites. In a search for novel agonist radioligands and imaging ligands for the α7 nicotinic receptor, [ 3H]AZ11637326 was synthesized and its in vitro membrane binding properties were characterized. [ 3H]AZ11637326 bound to hα7-HEK membranes with high specificity (> 95%), high affinity (230 pM) and a B max of 460 fmol/mg. The rank order of affinity of nicotinic standards determined with [ 3H]AZ11637326 strongly correlated with those determined using the classical α7 antagonist [ 125I]α-bungarotoxin, indicating that [ 3H]AZ11637326 bound to hα7-HEK membranes with an α7 nicotinic-like pharmacology. The K i values for the standards were on average 2.3-fold lower affinity than determined using the prototypical α7 nicotinic antagonist [ 125I]α-bungarotoxin. Because [ 3H]AZ11637326 specific binding is rapid and reversible, the K i values determined using this ligand are more accurate estimates of affinity than those determined using the kinetically sluggish [ 125I]α-bungarotoxin. [ 3H]AZ11637326 also bound to a high affinity (510 pM), nicotine-sensitive site on rat hippocampal membranes with an average B max of 55 fmol/mg. With rat hippocampal membranes, the nicotine-sensitive fraction of total binding was sub-optimal for a radioligand (~ 50%), yet the potencies and rank order of the K i values for standards were consistent with an α7 nicotinic pharmacology. Overall, these studies indicate that [ 3H]AZ11637326 is a useful new in vitro probe of the α7 nicotinic receptor agonist site and support its potential utility for in vivo receptor occupancy studies.

Scanning Mutagenesis of α-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the α9α10 Nicotinic Acetylcholine Receptor

Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the alpha9alpha10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp(5)-Arg(7) and Asp(11)-Ile(15), because changes at these positions resulted in the loss of activity at the alpha9alpha10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K+N9A, all of which were more potent than Vc1.1 at both the rat alpha9alpha10 and the human alpha9/rat alpha10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the alpha3beta2, alpha3beta4, and alpha7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the alpha9alpha10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the alpha9alpha10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.

Functional Signatures of α‐7 Nicotinic Acetylcholine Receptor Positive Allosteric Modulators: Effects on Channel Biophysics and Pharmacology.

Activation of neuronal α7 nicotinic acetylcholine receptors by agonists evokes rapidly activating, desensitizing currents, predominantly carried by Na+ and Ca2+ ions. Positive allosteric modulators (PAMs) of α7 nAChRs are devoid of intrinsic agonist activity but enhance α7 currents with or without effects on channel kinetics. Using patch clamp and Ca2+ flux assays in GH4C1 cells heterologoulsly expressing rat α7 receptors we describe the mechanism of action of α7 PAMs and their modulation by changes in temperature and repeat applications. All PAMs enhanced the current amplitude. Additionally the type‐2 PAMs also slowed channel desensitization. We show that PNU‐120596, a type‐2 PAM exhibited reduced potentiation of current amplitude upon repeated applications with no change in the EC50 of activation. PNU‐120596 also "dedesensitizes" receptors fully desensitized by exposure to high concentrations of agonists. NS‐1738, a type‐1 PAM exhibited a modest decrease in maximal current amplitude upon repeated application accompanied with 3‐fold increase in potency. SB‐206553 a type‐1/2 PAM did not exhibit differences in the extent of current amplitude potentiation or pharmacology upon repeated application. Modulatory effects of PAMs, though significant at room temperature, were attenuated near physiological temperatures. Furthermore, PNU‐120596 but not SB‐206553 largely removed the strong inward rectification usually exhibited by α7 nAChR receptor agonists.

Investigating the role of protein folding and assembly in cell-type dependent expression of α7 nicotinic receptors using a green fluorescent protein chimera

To test the hypothesis that cell-dependent expression of α7 receptors is due to differences in protein folding or assembly, we constructed a chimeric rat α7 subunit with green fluorescent protein (GFP) at the receptor C-terminal. Expression of α7-GFP in Xenopus oocytes resulted in currents that were indistinguishable from wild type receptors but were only 33% of control. 125I-α-bungarotoxin (αBGT) binding at the oocyte surface was reduced to 23% of wild type. Transfection of α7-GFP into GH4C1 cells produced fluorescence that was less intense than GFP alone, but showed significant α-BGT binding compared to transfection with GFP. In contrast, α7-GFP transfection in SH-EP1, HEK293 and CHO-CAR cells produced fluorescence without αBGT binding. Flow cytometry of cells transfected with α7-GFP indicated fluorescence in both SH-EP1 and GH4C1 cells, but surface toxin binding sites and sites immunoprecipitated using anti-GFP antibodies were undetectable in SH-EP1 cells, suggesting a problem in folding/assembly rather than trafficking. Surprisingly, integrated fluorescence intensities in GH4C1 cells transfected with α7-GFP did not correlate with amounts of cell surface or immunoprecipitable αBGT binding. Therefore, GFP folding at the C-terminal of the α7-GFP chimera is cell-line independent, but toxin binding is highly cell-line dependent, suggesting that if altered protein folding is involved in the cell-type dependence of α7 receptor expression, the phenomenon is restricted to specific protein domains. Further, C-terminal GFP-labeled α7 receptors decreased the efficiency of folding/assembly not only of chimeric subunits, but also wild-type subunits, suggesting that the C-terminal is an important domain for α7 receptor assembly.

Inhibition of Neuronal Nicotinic Acetylcholine Receptor Subtypes by α-Conotoxin GID and Analogues

alpha-Conotoxins are small disulfide-rich peptides from the venom of the Conus species that target the nicotinic acetylcholine receptor (nAChR). They are valuable pharmacological tools and also have potential therapeutic applications particularly for the treatment of chronic pain. alpha-Conotoxin GID is isolated from the venom of Conus geographus and has an unusual N-terminal tail sequence that has been shown to be important for binding to the alpha4beta2 subtype of the nAChR. To date, only four conotoxins that inhibit the alpha4beta2 subtype have been characterized, but they are of considerable interest as it is the most abundant nAChR subtype in the mammalian brain and has been implicated in a range of diseases. In this study, analysis of alanine-scan and truncation mutants of GID reveals that a conserved proline in alpha-conotoxins is important for activity at the alpha7, alpha3beta2, and alpha4beta2 subtypes. Although the proline residue was the most critical residue for activity at the alpha3beta2 subtype, Asp(3), Arg(12), and Asn(14) are also critical at the alpha7 subtype. Interestingly, very few of the mutations tested retained activity at the alpha4beta2 subtype indicating a tightly defined binding site. This lack of tolerance to sequence variation may explain the lack of selective ligands discovered for the alpha4beta2 subtype to date. Overall, our findings contribute to the understanding of the structure-activity relationships of alpha-conotoxins and may be beneficial for the ongoing attempts to exploit modulators of the neuronal nAChRs as therapeutic agents.

Dilinoleoylphosphatidylcholine ameliorates scopolamine-induced impairment of spatial learning and memory by targeting α7 nicotinic ACh receptors

Aims The present study was conducted to understand the role of 1,2-dilynoleoyl- sn-glycero-3-phosphocholine (DLPhtCho) in cognitive functions. Main methods Two-electrode voltage-clamp was made to Xenopus oocytes expressing rat α7 acetylcholine (ACh) receptors. Field excitatory postsynaptic potentials (fEPSPs) were monitored from the CA1 region of rat hippocampal slices. Water maze test was carried out to assess spatial learning and memory for rats. Key findings In the oocyte expression system, DLPhtCho at a concentration of 10 µM potentiated ACh-evoked currents to approximately 190% of basal amplitudes 70 min after 10-min treatment. In contrast, 1-stearoyl-2-lynoleoyl- sn-glycero-3-phosphocholine (SLPhtCho), 1-palmitoyl-2-linoleoyl- sn-glycero-3-phosphocholine (PLPhtCho), and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPhtCho) had no effect on the currents. DLPhtCho (10 µM) enhanced slope of fEPSPs to about 150% of basal levels at 70-min treatment, that is inhibited by α-bungarotoxin, an inhibitor of α7 ACh receptors, while no enhancement was obtained with SLPhtCho, PLPhtCho, or POPhtCho. In the water maze test, oral administration with DLPhtCho (5 mg/kg) significantly shortened the prolonged acquisition latency for rats intraperitoneally injected with scopolamine (1 mg/kg). Significance The results of the present study show that DLPhtCho improves scopolamine-induced learning and memory deficits, possibly by facilitating hippocampal synaptic transmission under the control of α7 ACh receptors. DLPhtCho, therefore, could be developed as a beneficial anti-dementia drug.

αConotoxin Arenatus IB[V11L,V16D] Is a Potent and Selective Antagonist at Rat and Human Native α7 Nicotinic Acetylcholine Receptors

A recently developed alpha-conotoxin, alpha-conotoxin Arenatus IB-[V11L,V16D] (alpha-CtxArIB[V11L,V16D]) [corrected], is a potent and selective competitive antagonist at rat recombinant alpha7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. alpha7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues, where they are implicated in a variety of functions. In this study, we evaluate this toxin at rat and human native nAChRs. Functional alpha7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 [1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea] in rat PC12 cells and human SH-SY5Y cells loaded with calcium indicators. alpha-CtxArIB[V11L,V16D] specifically inhibited alpha7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli, 5-I-A-85380 [5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride], nicotine, or KCl, that did not activate alpha7 nAChRs were unaffected. Human alpha7 nAChRs were also sensitive to alpha-CtxArIB[V11L, V16D]; acetylcholine-evoked currents in Xenopus laevis oocytes expressing human alpha7 nAChRs were inhibited by alpha-CtxArIB[V11L,V16D] (IC(50), 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the time course of recovery from blockade of rat alpha7 nAChRs in PC12 cells. alpha-CtxArIB[V11L,V16D] inhibited human native alpha7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 [(2)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin]-29-one] plus PNU-120596. Rat brain alpha7 nAChRs contribute to dopamine release from striatal minces; alpha-CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that alpha-CtxArIB[V11L,V16D] selectively inhibits human and rat native alpha7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating alpha7 nAChR functions.

Homology modeling and dynamics of the extracellular domain of rat and human neuronal nicotinic acetylcholine receptor subtypes α4β2 and α7

In recent years, it has become clear that the neuronal nicotinic acetylcholine receptor (nAChR) is a valid target in the treatment of a variety of diseases, including Alzheimer's disease, anxiety, and nicotine addiction. As with most membrane proteins, information on the three-dimensional (3D) structure of nAChR is limited to data from electron microscopy, at a resolution that makes the application of structure-based design approaches to develop specific ligands difficult. Based on a high-resolution crystal structure of AChBP, homology models of the extracellular domain of the neuronal rat and human nAChR subtypes alpha4beta2 and alpha7 (the subtypes most abundant in brain) were built, and their stability assessed with molecular dynamics (MD). All models built showed conformational stability over time, confirming the quality of the starting 3D model. Lipophilicity and electrostatic potential studies performed on the rat and human alpha4beta2 and alpha7 nicotinic models were compared to AChBP, revealing the importance of the hydrophobic aromatic pocket and the critical role of the alpha-subunit Trp-the homolog of AChBP-Trp 143-for ligand binding. The models presented provide a valuable framework for the structure-based design of specific alpha4beta2 nAChR subtype ligands aimed at improving therapeutic and diagnostic applications.

Aromatic residues at position 55 of rat α7 nicotinic acetylcholine receptors are critical for maintaining rapid desensitization

The rat alpha7 nicotinic acetylcholine receptor (nAChR) can undergo rapid onset of desensitization; however, the mechanisms of desensitization are largely unknown. The contribution of a tryptophan (W) residue at position 55 of the rat alpha7 nAChR subunit, which lies within the beta2 strand, was studied by mutating it to other hydrophobic and/or aromatic amino acids, followed by voltage-clamp experiments in Xenopus oocytes. When mutated to alanine, the alpha7-W55A nAChR desensitized more slowly, and recovered from desensitization more rapidly, than wildtype alpha7 nAChRs. The contribution of desensitization was validated by kinetic modelling. Mutating W55 to other aromatic residues (phenylalanine or tyrosine) had no significant effect on the kinetics of desensitization, whereas mutation to various hydrophobic residues (alanine, cysteine or valine) significantly decreased the rate of onset and increased the rate of recovery from desensitization. To gain insight into possible structural rearrangements during desensitization, we probed the accessibility of W55 by mutating W55 to cysteine (alpha7-W55C) and testing the ability of various sulfhydryl reagents to react with this cysteine. Several positively charged sulfhydryl reagents blocked ACh-induced responses for alpha7-W55C nAChRs, whereas a neutral sulfhydryl reagent potentiated responses; residue C55 was not accessible for modification in the desensitized state. These data suggest that W55 plays an important role in both the onset and recovery from desensitization in the rat alpha7 nAChR, and that aromatic residues at position 55 are critical for maintaining rapid desensitization. Furthermore, these data suggest that W55 may be a potential target for modulatory agents operating via hydrophobic interactions.

Broad-Spectrum Efficacy across Cognitive Domains by α7 Nicotinic Acetylcholine Receptor Agonism Correlates with Activation of ERK1/2 and CREB Phosphorylation Pathways

The alpha7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and may represent a drug target for treating cognitive deficits in neurodegenerative and psychiatric disorders. In the present study, we used a novel alpha7 nAChR-selective agonist, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) to interrogate cognitive efficacy, as well as examine potential cellular mechanisms of cognition. Exhibiting high affinity to native rat (Ki = 10.8 nM) and human (Ki = 16.7 nM) alpha7 nAChRs, A-582941 enhanced cognitive performance in behavioral assays including the monkey delayed matching-to-sample, rat social recognition, and mouse inhibitory avoidance models that capture domains of working memory, short-term recognition memory, and long-term memory consolidation, respectively. In addition, A-582941 normalized sensory gating deficits induced by the alpha7 nAChR antagonist methyllycaconitine in rats, and in DBA/2 mice that exhibit a natural sensory gating deficit. Examination of signaling pathways known to be involved in cognitive function revealed that alpha7 nAChR agonism increased extracellular-signal regulated kinase 1/2 (ERK1/2) phosphorylation in PC12 cells. Furthermore, increases in ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation were observed in mouse cingulate cortex and/or hippocampus after acute A-582941 administration producing plasma concentrations in the range of alpha7 binding affinities and behavioral efficacious doses. The MEK inhibitor SL327 completely blocked alpha7 agonist-evoked ERK1/2 phosphorylation. Our results demonstrate that alpha7 nAChR agonism can lead to broad-spectrum efficacy in animal models at doses that enhance ERK1/2 and CREB phosphorylation/activation and may represent a mechanism that offers potential to improve cognitive deficits associated with neurodegenerative and psychiatric diseases, such as Alzheimer's disease and schizophrenia.

In vitro screening strategies for nicotinic receptor ligands

A common historical strategy to the discovery of nicotinic receptor ligands has involved the use of radioligand-binding assays for ligand identification in combination with two-electrode voltage clamp in Xenopus oocytes for electrophysiological characterization. More recently, higher-throughput methodologies have replaced these approaches to accommodate screening of large compound libraries and to provide increased capacity for electrophysiological profiling in mammalian cell lines. We, and others, have implemented cell-based screening assays using the fluorometric imaging plate reader (FLIPR™) for primary and lead optimization screening of nicotinic receptor agonists and positive allosteric modulators (PAMs). Using GH4C1 cells expressing the rat α7 nicotinic receptor, both acetylcholine and nicotine produced concentration-dependent elevations of intracellular calcium with EC 50 values of 5.5 and 1.6 μM, respectively. PAM activity was robustly detected using the FLIPR assay; for example, the known α7 receptor PAM 5-hydroxyindole failed to directly activate the receptor but produced a leftward shift of the nicotine concentration–response curve in combination with a potentiation of the maximum evoked response to nicotine. Electrophysiological confirmation of agonist activity was achieved using the Dynaflow rapid perfusion system and patch clamp in the same GH4C1 cell expression system. Estimated EC 50 values for acetylcholine-evoked currents in GH4C1/α7 cells were 55 and 576 μM for area-under-the-curve (AUC) and maximum peak height calculations, respectively. Similarly, PAM activity was confirmed using electrophysiological recordings while also allowing for the mechanistic discrimination of compounds, not possible using the FLIPR assay. Specifically, PAMs capable of slowing the rapid desensitization of α7 receptors to different extents were discernable in these studies. Further improvements in the capacity to screen compounds using electrophysiology has been achieved by implementation of high-throughput gigaohm quality recording systems such as the QPatch and PatchXpress where agonist EC 50 values are highly comparable to those obtained using conventional manual patch clamp.

α7 Nicotinic Receptor Gene Promoter Polymorphisms in Inbred Mice Affect Expression in a Cell Type-specific Fashion

Inbred mouse strains display significant differences in their levels of brain alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) expression, as measured by binding of the alpha7-selective antagonist alpha-bungarotoxin. Variations in alpha-bungarotoxin binding have been shown to correlate with an animal's sensitivity to nicotine-induced seizures and sensory gating. In two inbred mouse strains, C3H/2Ibg (C3H) and DBA/2Ibg (DBA/2), the inter-strain binding differences are linked to a restriction length polymorphism in the alpha7 nAChR gene, Chrna7. Despite this finding, the molecular mechanism(s) through which genetic variability in Chrna7 may contribute to alpha7 nAChR expression differences remains unknown. However, studies of the human alpha7 nAChR gene (CHRNA7) previously have demonstrated that CHRNA7 promoter polymorphisms are associated with differences in promoter activity as well as differences in sensory processing. In the present study, a 947-base pair region of the Chrna7 promoter was cloned from both the C3H and DBA/2 inbred mouse strains in an attempt to identify polymorphisms that may underlie alpha7 nAChR differential expression. Sequence analysis of these fragments identified 14 single nucleotide polymorphisms (SNPs). A combination of two of these SNPs affects promoter activity in an in vitro luciferase reporter assay. These results suggest a mechanism through which the Chrna7 promoter genotype may influence interstrain variations in alpha7 nAChR expression.

Partial agonist and neuromodulatory activity of S 24795 for alpha7 nAChR responses of hippocampal interneurons

S 24795 evoked methyllycaconitine-sensitive inward currents in voltage-clamped hippocampal interneurons with maximum amplitude about 14% that of ACh-evoked responses. Experiments with rat α7 receptors expressed in Xenopus oocytes confirmed that S 24795 is a partial agonist of α7 nAChR with an EC 50 of 34 ± 11 μM and I max of approximately 10% relative to ACh. When 60 μM ACh was co-applied to α7-expressing oocytes along with increasing concentrations of S 24795, there was a progressive decrease in response compared to the responses to 60 μM ACh alone (IC 50 45 ± 9 μM). The positive allosteric modulator 5-hydroxyindole potentiated ACh- and S 24795-evoked responses of α7 receptors in both oocytes and hippocampal interneurons. In hippocampal slice experiments, depending on the ACh concentrations in the application pipette and the ratio of ACh to S 24795, co-application of S 24795 with ACh variously increased, decreased, or had no effect on responses, compared to ACh alone. In order to estimate the effective dilution factor for the pressure application experiments, we tested α7 receptors in oocytes with ACh alone and in co-application with S 24795 at the same ratios as in the slice experiments, but at varying dilution factors. The pattern of interaction seen in the slice experiments was most closely matched under the conditions of a 3:100 dilution, suggesting that the pipette solution was diluted approximately 30-fold at the site of action. This dilution factor was consistent with the potency of ACh and S 24795 in the oocyte expression system (EC 50s ≈30 μM).

The Novel α7 Nicotinic Acetylcholine Receptor AgonistN-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-7-[2-(methoxy)phenyl]-1-benzofuran-2-carboxamide Improves Working and Recognition Memory in Rodents

The relative contribution of alpha4beta2, alpha7 and other nicotinic acetylcholine receptor (nAChR) subtypes to the memory enhancing versus the addictive effects of nicotine is the subject of ongoing debate. In the present study, we characterized the pharmacological and behavioral properties of the alpha7 nAChR agonist N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-7-[2-(methoxy)phenyl]-1-benzofuran-2-carboxamide (ABBF). ABBF bound to alpha7 nAChR in rat brain membranes (Ki=62 nM) and to recombinant human 5-hydroxytryptamine (5-HT)3 receptors (Ki=60 nM). ABBF was a potent agonist at the recombinant rat and human alpha7 nAChR expressed in Xenopus oocytes, but it did not show agonist activity at other nAChR subtypes. ABBF acted as an antagonist of the 5-HT3 receptor and alpha3beta4, alpha4beta2, and muscle nAChRs (at higher concentrations). ABBF improved social recognition memory in rats (0.3-1 mg/kg p.o.). This improvement was blocked by intracerebroventricular administration of the alpha7 nAChR antagonist methyllycaconitine at 10 microg, indicating that it is mediated by alpha7 nAChR agonism. In addition, ABBF improved working memory of aged rats in a water maze repeated acquisition paradigm (1 mg/kg p.o.) and object recognition memory in mice (0.3-1 mg/kg p.o.). Rats trained to discriminate nicotine (0.4 mg/kg s.c.) from vehicle did not generalize to ABBF (0.3-30 mg/kg p.o.), suggesting that the nicotine cue is not mediated by the alpha7 nAChR and that selective alpha7 nAChR agonists may not share the abuse liability of nicotine. Our results support the hypothesis that alpha7 nAChR agonists may provide a novel therapeutic strategy for the treatment of cognitive deficits with low abuse potential.

Detection of α7 nicotinic acetylcholine receptors with the aid of antibodies and toxins

Neuronal nicotinic acetylcholine receptors (nAChRs) containing α7 subunit are well represented in the brain and some non-neuronal tissues, and their malfunctioning is associated with diverse pathologies. Therefore, detection and quantification of α7 nAChR are important tasks. The affinity-purified antibodies were prepared against the 1–23 and 179–190 fragments of the human and rat α7 nAChR extracellular domain. The specificity and selectivity of these α7 (1–23) and α7 (179–190) antibodies was tested by ELISA in model systems: the E. coli-expressed α7 subunit extracellular domain and the pituitary cell line GH4C1 stably expressing human α7 nAChR. On the rat brain slices two antibodies and biotinylated α-cobratoxin specifically stained the hippocampus region known to be rich in α7 nAChR. Western blot analysis revealed that in the human thalamus membranes and in rat brain membranes, antibodies α7 (1–23) stained a single band of 62 kDa, while the α7 (179–190) antibodies stained a doublet of 53–54 kDa. The results obtained show that utilization of model systems and a combination of several antibodies with appropriately labeled toxins may provide better ways for detection of α7 nAChR.

α7 Nicotinic receptor gene delivery into mouse hippocampal neurons leads to functional receptor expression, improved spatial memory-related performance, and tau hyperphosphorylation

Brain α7 nicotinic receptors have become therapeutic targets for Alzheimer’s disease (AD) based on their memory-enhancing and neuroprotective actions. This study investigated the feasibility of increasing neuronal α7 receptor functions using a gene delivery approach based on neuron-selective recombinant adeno-associated virus (rAAV)–derived vectors. In order to determine whether α7 receptor-mediated cytotoxicity was dependent on receptor density, rat α7 nicotinic receptors were expressed at high concentrations in GH4C1 cells as measured with nicotine-displaceable [3H]methyllycaconitine (MLA) binding. The potency of GTS-21 (an α7 receptor agonist) to induce cell loss was similar in these cells to that seen in pheochromocytoma (PC12) cells expressing nine-times-lower receptor levels, suggesting that cytotoxicity was more dependent on agonist concentration than receptor density. Hippocampal transduction with rat α7 nicotinic receptors increased [3H]MLA binding in this region in wild type and α7 receptor-knockout (KO) mice without apparent cytotoxicity. No difference was observed in Kd values for MLA binding between endogenous and transgenic receptors. Single cell recordings demonstrated that dentate granule cells that normally have no α7 receptor response did so following α7 receptor gene delivery in wild type mice. Recovery of α7 function was also observed in stratum oriens and stratum radiatum neurons of KO mice following gene delivery. Wild type mice exhibited improved acquisition performance in the Morris water task 1 month after bilateral hippocampal transductions with the rat α7 receptor gene compared with green fluorescent protein–transduced controls. However, both groups reached similar training levels and there was no difference in subsequent probe performance. Finally, this gene delivery approach was used to test whether α7 receptors affect tau-phosphorylation. Chronic (i.e. 2 month but not 2 week) expression of high levels of α7 receptors in hippocampus increased AT8 staining characteristic of hyperphosphorylated tau in that region, indicating that endogenous agonist-mediated receptor activation may be able to modulate this process.