Targeting α7 neuronal acetylcholine receptors (nAChRs) with selective agonists and positive allosteric modulators (PAMs) is considered a therapeutic approach for managing cognitive deficits in schizophrenia and Alzheimer's disease. In this study, we describe a novel type II α7 PAM, 4-(5-(4-chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744), that exhibits a unique pharmacological profile. In oocytes expressing α7 nAChRs, A-867744 potentiated acetylcholine (ACh)-evoked currents, with an EC50 value of ∼1 μM. At highest concentrations of A-867744 tested, ACh-evoked currents were essentially nondecaying. At lower concentrations, no evidence of a distinct secondary component was evident in contrast to 4-naphthalen-1-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonic acid amide (TQS), another type II α7 PAM. In the presence of A-867744, ACh concentration responses were potentiated by increases in potency, Hill slope, and maximal efficacy. When examined in rat hippocampus CA1 stratum radiatum interneurons or dentate gyrus granule cells, A-867744 (10 μM) increased choline-evoked α7 currents and recovery from inhibition/desensitization, and enhanced spontaneous inhibitory postsynaptic current activity. A-867744, like other α7 PAMs tested [1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)urea (NS1738), TQS, and 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596)], did not displace the binding of [3H]methyllycaconitine to rat cortex α7* nAChRs. However, unlike these PAMs, A-867744 displaced the binding of the agonist [3H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539) in rat cortex, with a Ki value of 23 nM. A-867744 neither increased agonist-evoked responses nor displaced the binding of [3H]A-585539 in an α7/5-hydroxytryptamine3 (α7/5-HT3) chimera, suggesting an interaction distinct from the α7 N terminus or M2-3 loop. In addition, A-867744 failed to potentiate responses mediated by 5-HT3A or α3β4 and α4β2 nAChRs. In summary, this study identifies a novel and selective α7 PAM showing activity at recombinant and native α7 nAChRs exhibiting a unique pharmacological interaction with the receptor.
Neuronal nicotinic acetylcholine receptors (nAChRs) belong to the pentameric superfamily of ligand-gated ion channels that includes 5-HT3, GABAA/C, and glycine receptors. These receptors are composed of either homomeric α or heteromeric α/β subunit combinations. Currently, 12 neuronal nicotinic subunits have been identified (α2–α10; β2–β4) (Paterson and Nordberg, 2000; Gotti et al., 2006). One subtype abundantly expressed in the central nervous system, including in regions involved in learning and memory (hippocampus and cerebral cortex), is the homomeric α7 subunit (Rubboli et al., 1994; Wevers et al., 1994). Homomeric α7 nAChRs, when expressed in heterologous expression systems, activate and desensitize rapidly, and they exhibit relatively higher calcium permeability compared with other nAChR combinations (Sands et al., 1993; Dajas-Bailador and Wonnacott, 2004). This increased Ca2+ permeability is thought to stimulate downstream events, including extracellular signal-regulated kinase and cAMP response element-binding protein pathways involved in learning and cognition (Sweatt, 2004; Bitner et al., 2007). In addition, activation of α7 nAChRs contributes to neuronal excitability (Frazier et al., 1998), modulates the release of excitatory and inhibitory neurotransmitters (Alkondon et al., 2000), exhibits neuroprotective effects in experimental in vitro models of cellular damage (Levin and Rezvani, 2002), and shows procognitive effects in in vivo animals models of learning and memory (Cincotta et al., 2008).
Activation or enhancement of α7 nAChR function can occur via either direct agonist activation of the orthosteric site or via positive allosteric modulation. The recent focus on the latter mechanism has led to the identification of structurally diverse positive allosteric modulators (PAMs), including SB-206553 (Dunlop et al., 2009), PNU-120596 (Hurst et al., 2005), TQS (Grønlien et al., 2007), CCMI (also referred as compound 6/Q or XY4083) (Ng et al., 2007), and NS1738 (Timmermann et al., 2007). In addition, genistein (Grønlien et al., 2007), galantamine (Samochocki et al., 2003), bovine serum albumin (Conroy et al., 2003), SLURP-1 (Chimienti et al., 2003), an acetylcholinesterase-derived peptide (Zbarsky et al., 2004), and (2-amino-5-keto)thiazole compounds (Broad et al., 2006) also function as PAMs. Based on biophysical properties at least two different profiles of α7 PAMs are distinguished: type I— exemplified by 5-HI and genistein— predominantly increasing peak current amplitude response, and type II—represented by PNU-120596 and TQS—affecting both peak current response and current decay profile (Bertrand and Gopalakrishnan, 2007; Grønlien et al., 2007). Both type I and II PAMs have been shown to exhibit efficacy in vivo behavioral models. For example, PNU-120596 reversed amphetamine-induced gating deficits in rats, and CCMI improved gating deficits in DBA/2 mice (Hurst et al., 2005; Ng et al., 2007). These observations show that α7 PAMs, belonging to both types, are effective in certain preclinical in vivo models. The differential spectrum of behavioral efficacy relevant to cognitive deficits in Alzheimer's disease and schizophrenia remains to be fully elucidated.
The mechanisms or sites by which PAMs interact with α7 nAChRs to enhance the receptor function are incompletely understood. Emerging structure-function studies using chimeric and single amino acid point mutation approaches have suggested distinct regions responsible for the PAM effects of NS1738 and PNU-120596, respectively, in the N terminus and transmembrane domains (Bertrand et al., 2008; Young et al., 2008). Given the structural diversity of α7 PAM chemotypes, it is likely that there are multiple allosteric sites on the receptor. Thus, the α7 PAM interaction with the nAChRs may be similar to PAM binding to GABAA receptors. At the GABAA receptors, three different allosteric sites have been proposed: 1) within the ion channel domain transmembrane 2, near the extracellular end; 2) in the proximity of the agonist binding sites; and 3) in the linker region stretching from the agonist site loop C to the top of the transmembrane 1 region (Olsen et al., 2004).
In this study, we describe A-867744, as a novel type II PAM. We demonstrate that this compound selectively potentiates α7 but not α4β2, α3β4, or 5-HT3A receptors and that it displays unique pharmacology based on radioligand and functional studies distinguishing this compound from other known α7 PAMs.
Materials and Methods
Materials. Oocytes were obtained from adult female Xenopus laevis frogs (Blades Biological Ltd., Cowden, Edenbridge, Kent, UK) and male Sprague-Dawley rats (10–40 days old) from Charles River Breeding Laboratories (Portage, MI). Animals were cared for in accordance with the Institutional Animal Care Committee guidelines that meet the guidelines of the National Institutes of Health (Bethesda, MD). Acetylcholine, choline, methyllycaconitine (MLA), and nicotine were obtained from Sigma-Aldrich (St. Louis, MO or Oslo, Norway) or from Tocris Bioscience (Ellisville, MO or Bristol, UK). PNU-120596, TQS, PNU-282987, and [3H]A-585539 were synthesized at Abbott (Abbott Park, IL). A-867744 was also synthesized at Abbott and dissolved in dimethyl sulfoxide as stock solution (10 or 30 mM) and diluted to appropriate test concentrations right before experiments to prevent the compound from precipitating out, especially at higher concentrations tested after extended preincubation. NS6784 was obtained from NeuroSearch (Ballerup, Denmark). [3H]MLA, TTX, APV, CNQX, picrotoxin, 5-HT, and atropine were obtained from Tocris Bioscience or Sigma-Aldrich. All other chemicals and reagents were obtained from Sigma-Aldrich or Fisher Scientific (Essex, UK).
Two-Electrode Voltage Clamp in X. laevis Oocytes.X. laevis oocytes were prepared for electrophysiological experiments as described previously (Grønlien et al., 2007; Briggs et al., 2008). In brief, three to four lobes from ovaries of female adult X. laevis frogs were removed, manually defolliculated, and treated with collagenase type 1A (2 mg/ml; Sigma-Aldrich) prepared in low-Ca2+ Barth's solution [90 mM NaCl, 1.0 mM KCl, 0.66 mM NaNO3, 2.4 mM NaHCO3, 10 mM HEPES, 2.5 mM sodium pyruvate, 0.82 mM MgCl2, and 0.5% (v/v) penicillin-streptomycin solution purchased from Sigma-Aldrich, pH 7.55] for 1.5 to 2 h at ∼18°C under constant agitation to obtain isolated oocytes. The oocytes were injected with ∼20 to 25 ng of human or rat α7 nAChR cRNA, or ∼1 to 5 ng of human 5-HT3A cRNA, kept at 18°C in a humidified incubator in modified Barth's solution [90 mM NaCl, 1.0 mM KCl, 0.66 mM NaNO3, 2.4 mM NaHCO3, 10 mM HEPES, 2.5 mM sodium pyruvate, 0.74 mM CaCl2, 0.82 mM MgCl2, 0.5% (v/v) penicillin-streptomycin solution, pH 7.55] and used 2 to 7 days after injection. Responses were measured by two-electrode voltage-clamp technique using Parallel Oocyte Electrophysiology Test station (Trumbull et al., 2003). During recordings, the oocytes were bathed in Ba2+-OR2 solution (90 mM NaCl, 2.5 mM KCl, 2.5 mM BaCl2, 1.0 mM MgCl2, 5.0 mM HEPES, and 0.0005 mM atropine, pH 7.4) and held at -60 mV at room temperature (∼20°C). Agonists were applied to recording chambers at ∼6 ml/s as indicated. Modulator test compounds were applied for a minimum of 60 s before agonist application, allowing for sufficient preincubation unless otherwise stated. Agonist application after preincubation with modulator was always done in the presence of the test modulator.
Calcium and FMP Imaging. Functional nAChR activities were assessed in HEK-293 cells expressing human α4β2 or α3β4 subunits and in IMR-32 cells by measuring intracellular calcium changes using Fluo-4 acetoxymethyl ester or no-wash calcium dye (Applied Biosystems/MDS Analytical Technologies/Molecular Devices, Sunnyvale, CA or Invitrogen, Carlsbad, CA) and the fluorometric imaging plate reader (FLIPR) system (Applied Biosystems/MDS Analytical Technologies/Molecular Devices) as described previously (Grønlien et al., 2007; Briggs et al., 2008; Gopalakrishnan et al., 2008; Faghih et al., 2009). The responses in HEK-293 cells expressing α7/5-HT3 chimera (Bertrand et al., 2008) were determined using FMP dye (Molecular Devices). In brief, cells were plated at densities of 25,000 to 100,000 cells/well in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and appropriate antibiotic selection (HEK-293-derived cell lines) or minimum essential media supplemented with 10% fetal bovine serum and 1 mM sodium pyruvate, 1% nonessential amino acids, and 1% antibiotic-antimycotic solution (IMR-32 cells) in 96-well clear-bottomed black walled plates precoated with poly-d-lysine and allowed to incubate for 24 to 48 h at 37°C in 5% CO2 in a humidified environment. After aspirating the media, cells were incubated for ∼45 to 60 min with Fluo-4 acetoxymethyl ester, no-wash calcium dye, or FMP indicator dye in the dark at room temperature. Calcium dyes were dissolved in N-methyl-d-glucamine/Ringer's buffer (140 mM N-methyl-d-glucamine, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES, and 10 mM CaCl2, pH 7.4), whereas FMP dye was dissolved in Ca2+- and Mg2+-free phosphate-buffered saline supplemented with 0.1 mM CaCl2, 0.1 mM MgCl2, and 10 mM HEPES. When required after dye loading, cells were gently washed with the same buffer, removing extracellular dye and leaving ∼100 μl/well after the final wash. The protocol used in studies involving HEK-293-derived cell lines was as follows. Cells were placed in the FLIPR chamber where 50 μl of 3× stock concentration of test modulators or buffer prepared in the loading buffer was applied in the first addition for 5 min. In the second addition also for 5 min, 50 μl of 4× stock concentrations of agonist (nicotine or PNU-282987) or buffer was added. In IMR-32 cells, double addition protocol was also used. To determine the concentration responses of A-867744, various concentrations of A-867744 were given in the first addition followed by 1 μM NS6784 in the second application. In another set of experiments described in Fig. 3 (reactivation by A-867744), NS6784 (1 μM) was applied first, followed by MLA (100 or 300 nM) or blank, and last by A-867744 (10 μM). All experiments were carried out at room temperature (∼20–22°C).
Whole-Cell Patch-Clamp Recordings in Brain Slices. Hippocampal brain slices were prepared from male Sprague-Dawley rats, fully anesthetized with Ultane (sevoflurane) or with CO2 gas, sacrificed by decapitation. Brain was rapidly removed and placed into ice-cold either high-Mg2+ artificial cerebral spinal fluid (ACSF: 130 mM NaCl, 2.8 mM KCl, 11.3 mM MgCl2, 2.5 mM CaCl2, 1.25 mM NaH2PO4, 10 mM dextrose, and 26 mM NaHCO3 gassed continuously with 95% O2, 5% CO2, pH 7.3–7.4 at ambient temperature) or regular Mg2+ ACSF (1 mM MgCl2 used instead of 11.3 mM). Hippocampal brain slices (250–400 μm in thickness, coronal or 30° parahorizontal) were prepared using standard procedures and cut at 1 to 3°C using a Vibratome slicer with temperature-controlled oxygenated ACSF bath. Slices were preincubated at 32°C for at least 1 h before use. For each experiment, one slice was selected, placed in a chamber perfused with ACSF at ambient temperature (∼22°C), and visualized using an E600FN microscope (Nikon, Tokyo, Japan) with infrared differential interference contrast optics. Whole-cell patch-clamp recordings were obtained from either hippocampus CA1 interneurons (stratum radiatum) to access fast somatic α7 currents or from hilar dentate gyrus granule cells to measure spontaneous inhibitory postsynaptic currents (IPSCs). Borosilicate glass capillary pipettes were filled with internal solution containing 10 mM CsCl, 0.5 mM CaCl2, 1 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 2 mM Mg-ATP, 0.3 mM Na-GTP, and 135 mM methane-sulfonic acid, adjusted to pH 7.3 with CsOH. Whole-cell recording was established, and neuronal activity was validated using voltage steps to activate characteristic sodium currents. IPSCs, presumably mediated by GABAA transmission, were recorded at 0 mV so that inward cationic currents (e.g., glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid excitatory postsynaptic currents) would be minimized and anionic currents (GABAA IPSCs) would be outward. IPSCs occurring spontaneously during 5-min epochs were recorded using a MultiClamp 700A amplifier, Digidata 1322A converter, and pClamp 9 software (Axon Instruments, Foster City, CA). When recordings from CA1 interneurons were made, cells were held at -80 mV in the presence of TTX (0.5 μM), APV (20–50 μM), CNQX (20–50 μM), picrotoxin (50 μM), and atropine (0.5 μM) added to the perfusing bath ACSF to minimize the contribution of neuronally mediated glutamatergic, GABAergic, and muscarinic activities. In the presence of the blockers, α7 agonist puffs—10 mM choline or 3 mM ACh in application pipette—were made using a Picospritzer II apparatus (General Valve, Fairfield, NJ) for 5- to 50-ms duration and 10 to 30 psi pressure setting. Puff pipettes were positioned 5 to 20 μm away from recording cells. The exact concentration of compound reaching the cells to evoke α7 currents cannot be precisely determined, but it would be expected to be considerably lower than in the puff pipette. In dentate gyrus experiments, compounds were applied by bath perfusion (1.8 ml/min flow rate; 600-μl chamber). Data were stored in a PC and analyzed using pClamp 9 or MiniAnalysis 6.0.3 software (Synaptsoft, Decatur, GA) as appropriate.
Whole-Cell Patch-Clamp Recordings in HEK-293 Cells Expressing α7/5-HT3 Chimera. Standard whole-cell patch-clamp technique was used to record from HEK-293 cells stably expressing chimeric α7/5-HT3 receptors (i.e., chimera 2 in Bertrand et al., 2008). Cells were plated 48 to 96 h on round poly-d-lysine-precoated glass coverslips before recordings. Experiments were done using pClamp 8.0 or 9.0 (Applied Biosystems/MDS Analytical Technologies/Axon Instruments) installed on a Dell Pentium III computer connected to an Axon 200B amplifier (Applied Biosystems/MDS Analytical Technologies/Axon Instruments, Sunnyvale, CA). Changes in test solution conditions were obtained using a pulled theta tube controlled by Perfusion Fast-Step system (Warner Instruments, Hamden, CT). The pipette solution contained 120 mM KCl, 30 mM NaCl, 2 mM MgCl2, 5 mM EGTA, and 10 mM HEPES, pH 7.2 to 7.3. The bath solution contained 140 mM NaCl, 5 mM KCl, 0.1 mM CalCl2, 0.1 mM MgCl2, 10 mM HEPES, and 0.5 μM atropine. Cells were held at -80 mV unless specified otherwise.
Radioligand Binding Experiments. Experiments were carried out as described previously (Anderson et al., 2008). Membrane homogenates were prepared from rat cortex and HEK-293 cells stably expressing chimera (i.e., chimera 2 in Bertrand et al., 2008). In ligand saturation experiments, [3H]A-585539 and [3H]MLA were used in concentrations ranging from 0.04 to 6 nM. In competition experiments, compounds were tested in increasing concentrations up to the highest of 30 μM, for their abilities to displace [3H]A-585539 or [3H]MLA binding. Nonspecific binding was defined with 10 or 30 μM MLA.
Data Analysis. In two-electrode voltage-clamp studies, responses were quantified by measuring peak current amplitude. Peak current responses were expressed as percentage response to 100 μM ACh (α7 currents) or 2 to 3 μM 5-HT (5-HT3A currents) in the presence of varying test modulator concentration responses, or to 1 mM ACh (without PAM) in agonist concentration responses (α7 oocytes). In Ca2+ imaging or FMP studies, raw fluorescence responses were corrected by subtracting fluorescence values from wells with buffer only added. Peak fluorescent responses were determined over the range of drug exposure using FLIPR software and expressed as a percentage of the reference peak response for 3 to 10 μM nicotine (α4β2 and α3β4) or 25 nM PNU-282987 (chimera studies) to determine the effects of A-867744. In FMP imaging studies, concentration-response experiments to PNU-292987 were normalized to the maximal control agonist response. Whole-cell patch-clamp responses determined in hippocampus brain slice were normalized to the choline-evoked responses obtained before the addition of A-867744. Radioligand binding Ki constants were calculated as described previously (Anderson et al., 2008). Concentration-response graphs were prepared using Prism (GraphPad Software Inc., San Diego, CA). Data are reported as means with 95% confidence intervals or S.E.M.), n is the number of independent determinations made, and p < 0.05 was considered statistically significant.
Potentiation of Recombinant Human α7 Currents. A-867744 is a representative and optimized compound from a series of pyrrole analogs functioning as positive allosteric modulators of α7 nAChRs (Faghih et al., 2009). In initial experiments, the effect of A-867744 was examined as an agonist at human α7 nAChRs. This compound up to the highest concentration tested of 30 μM did not evoke any α7 current. Higher concentrations of A-867744 could not be tested due to solubility limitation. When A-867744 was preincubated and α7 currents evoked by constant 100 μM ACh, concentration-dependent potentiation was noted, with a potency (EC50 value) of ∼1 μM and maximal efficacy of ∼730% or 7.3-fold (Figs. 1b and 2a; Table 1). As shown in Table 1, the responses at rat α7 nAChRs expressed in X. laevis oocytes were comparable with those identified at human α7 nAChRs, illustrating no interspecies differences in the ability of A-867744 to function as an α7 PAM. Figure 1b illustrates that A-867744 not only had a strong effect on the peak current response but also affected the decay of the current response that seemed concentration-dependent. In the presence of highest concentration tested (e.g., 3 μM A-867744), ACh-evoked currents were essentially nondecaying. Figure 1c depicts representative current responses obtained for another α7 PAM, TQS, whose properties were described in detail recently (Grønlien et al., 2007). Although at the higher concentrations tested, both compounds produced similar effects. At lower concentrations, there was a qualitative difference. TQS, but not A-867744, was able to evoke a secondary component with onset distinct from that of the initial current component. This suggests that the underlying mechanisms responsible for modulation by these two PAMs are probably different.
Next, we examined antagonist sensitivity of the current responses to MLA, an α7 nAChR antagonist. As shown by Fig. 1d, pretreatment with MLA abolished A-867744 potentiated ACh-evoked current responses. This supports interaction of A-867744 with α7 nAChRs.
We have further determined the effect of A-867744 on ACh concentration responses. As shown in Fig. 2b, in the presence of A-867744 (tested at nearly fully efficacious PAM concentration of 5 μM), ACh responses were potentiated as reflected by increases in potency (from ∼150 to 40 μM), maximal efficacy (from ∼110 to 500%), and Hill slope values (∼1.5 to 3.5) (Fig. 2b).
Reactivation of Desensitized α7 nAChRs by A-867744. Previously, we and others have shown that type II PAMs, specifically TQS and PNU-120596, were able to reactivate desensitized α7 currents (Hurst et al., 2005; Grønlien et al., 2007). In this study, we examined whether A-867744 exhibits a similar effect. α7 nAChR currents were evoked and desensitized by continued presence of ACh, and A-867744 was applied in the presence of ACh (Fig. 1e). As shown, A-867744 was able to reactivate the desensitized currents, showing a nondecaying steady-state nature. When normalized to 1 mM ACh (without A-867744), the maximal A-867744 reactivated currents were 29.3 ± 11.3% (n = 4) and 59.5 ± 2.0% (n = 5) for 3 and 10 μM PAM, respectively. We also investigated the reactivation of desensitized α7 nAChRs by A-867744 in IMR-32 cells using Ca2+ imaging. As shown in Table 1 and Fig. 3, the addition of a selective α7 agonist (NS6784) in the presence of A-867744 evoked Ca2+ transients; in comparison, the application of either A-867744 or α7 agonist (NS6784) alone had no effect on basal Ca2+. When the order was such, the agonist was given first at high concentrations, sufficient to desensitize the α7 nAChRs, followed by A-867744, Ca2+ transients were evoked. These A-867744-evoked responses were attenuated or reduced in the presence of 100 and 300 nM MLA by 62 ± 10% (n = 10; p < 0.001) and 90 ± 10% (n = 4; p < 0.01), respectively (Fig. 3), illustrating their dependence on the α7 nAChRs.
Selectivity of A-867744. As summarized in Table 1 and Fig. 2a, A-867744 potentiated responses mediated by α7 nAChRs, with an EC50 value of ∼1 to 2 μM as determined by electrophysiology and using Ca2+ imaging in IMR-32 cells endogenously expressing human α7 nAChRs. To determine whether A-867744 exhibits selectivity at α7 nAChRs, the effects of this compound were examined at two separate nAChR subtypes (human α4β2 and α3β4) and at human 5-HT3A. A-867744 did not activate directly as an agonist either α4β2 or α3β4 receptor-mediated responses up to the maximal concentration tested of 30 μM. In the presence of A-867744, the submaximal nicotine-evoked responses were not enhanced but rather inhibited. The IC50 value and maximal inhibition values were ∼6 μM and 96% and 20 μM and 64% at α4β2 or α3β4 receptors, respectively (Fig. 4; Table 1). These experiments illustrate that A-867744 is probably either a noncompetitive antagonist or negative allosteric modulator. Experiments were also carried out on human 5-HT3A currents expressed in X. laevis oocytes. A-867744 did not activate any 5-HT3 current up to the maximal concentration tested of 30 μM. In the presence of A-867744 (0.1–30 μM), the responses to submaximal 5-HT (2–3 μM) were unaffected illustrating lack of modulator activity at this receptor (Fig. 4).
Lack of Potentiation of α7/5-HT3 Chimera Responses by A-867744. Chimeric α7/5-HT3 receptors offer a unique opportunity to potentially identify domains of the α7 nAChR that influence allosteric modulator properties. We used a chimera derived from human α7 (N terminus and M2-3 loop) fused with human 5-HT3A receptor (rest), as described previously (Bertrand et al., 2008), and we expressed the chimeric receptor stably in HEK-293 cells. Both ACh (Fig. 5a) and choline were able to evoke the chimeric currents. When normalized to 3 mM ACh (100%), the addition of 3 mM choline and 30 μM ACh produced responses of 74 ± 5% (n = 4) and 46 ± 6% (n = 5), respectively. Consequently, the submaximal concentration of 30 μM ACh was used to investigate the effect of A-867744. A-867744 at 10 μM did not evoke any current, when added directed, and it failed to potentiate the chimeric currents evoked by 30 μM ACh. In fact, a modest degree of inhibition (∼50%) was noted (Fig. 5, a and b). The effect of A-867744 was not easily reversible.
Effects to A-867744 were also examined by measuring fluorescence changes in membrane potential (FMP dye imaging) in HEK-293 cells expressing chimeric α7/5-HT3A receptors evoked by the α7 agonist PNU-282987. As depicted in Fig. 5d, PNU-282987 concentration-dependently increased FMP fluorescence, with an EC50 value of ∼30 nM. Consequently, the submaximal concentration of 25 nM of this agonist was chosen to determine the effect of pretreatment with varying concentrations of A-867744. Figure 5a, consistent with the electrophysiological observation, shows that A-867744 failed to increase the signals. In addition, the comparison of the concentration responses to PNU-282987 measured in the presence or absence of 10 μM A-867744 revealed overlapping curves (Fig. 5b). In comparison with A-867744, NS1738 at 30 μM was able to potentiate the submaximal α7 agonist (25 nM PNU-282987)-evoked responses by 162.2 ± 1.6% (n = 2), illustrating that only certain PAMs are effective. These observations collectively demonstrate that the N-terminal region and extracellular loop M2-3 of the α7 nAChR are not obligatory for the positive allosteric modulator effect of A-867744.
Selective Displacement of Radiolabeled α7 nAChR Ligand by A-867744. A-867744 did not displace the binding of [3H]MLA (Fig. 6; Table 1) or [3H]cytisine (data not shown) in rat brain homogenates up to the highest concentration tested of 10 μM. [3H]A-585539 is an agonist that binds with high affinity to α7 nAChRs (Anderson et al., 2008). When tested in rat homogenates, A-867744 concentration-dependently displaced the binding of [3H]A-585539, with a Ki value of 0.023 μM and 70% maximal displacement as did nicotine and MLA. In contrast, the other α7 PAMs tested (PNU-120596, TQS, or 5-HI) did not displace the binding of [3H]A-585539 (Fig. 6). To further examine the effect of A-867744 on the displacement of [3H]A-585539, experiments were conducted on homogenates prepared from HEK-293 cells stably expressing chimeric human α7/5-HT3 receptors. [3H]A-585539 bound specifically to these chimeric receptors to a single affinity site, with Kd value of 0.151 ± 0.022 nM (n = 4). Unlike wild-type α7, A-867744 did not displace the binding of [3H]A-585539 in the chimera. TQS, NS1738, and 5-HI were also ineffective in displacing [3H]A-585539 in the chimera, whereas MLA and nicotine did so effectively (Fig. 6). This supports the notion that A-867744 interacts with a region of the nAChR distinct from the N-terminal domain, which contains the agonist/competitive antagonist binding site.
Effect of A-867744 on Native Rat α7 Currents in Hippocampus Brain Slices. Consistent with previous observations (Frazier et al., 1998; McQuiston and Madison, 1999), rapid application of ACh (3 mM) or choline (10 mM) evoked α7-like currents in rat hippocampus CA1 interneurons. The bath application of MLA (10 nM) completely inhibited these currents (data not shown), confirming the involvement of α7 nAChRs in the generation of these evoked responses.
To characterize the effect of A-867744 on native α7 currents, choline (10 mM in pipette) was puffed onto CA1 interneurons to evoke α7 currents and A-867744 (10 μM) supplied by bath application. A-867744 increased the peak current amplitude and current density (by ∼2.0-fold), total charge transfer (∼3.4-fold), half-width (∼3.3-fold), and current decay tau (∼2.5-fold). A-867744, hence, had robust effects on the evoked native α7 current kinetics (Fig. 7; Table 2).
The effect of A-867744 was also examined on the time course on the recovery of α7 currents using variable interval agonist puff applications (i.e., ratio of P2/P1). As shown in Fig. 7c, without A-867744, ∼15 s was required for the current to recover completely from desensitization. In the presence of 10 μM A-867744, a leftward shift in the recovery time course was observed. For example, at the 5-s interval in the presence of A-867744, α7 current recovered by ∼75%, whereas in the absence of the PAM this value was ∼40%. This observation demonstrates that A-867744 enhances the rate of the recovery of α7 current from desensitization/inhibition.
Potentiation of Inhibitory Synaptic Activity by A-867744 in Dentate Gyrus Granule Cells. α7 nAChRs are also present in dentate gyrus in which they are known to regulate GABAergic spontaneous IPSCs recorded in granule cells (Frazier et al., 2003). In this study, spontaneous IPSCs were also recorded from rat dentate gyrus granule cells, and the effect of A-867744 was determined. A representative experiment from this series of studies is shown in Fig. 8. The bath application of choline (1 mM) moderately increased the IPSC activity. The application of A-867744 (10 μM) that on its own did not have any effect, potentiated the choline evoked spontaneous IPSCs evoked over the range from ∼5 to 30 pA. This illustrates that A-867744 enhances the choline evoked GABAergic activity in the dentate gyrus.
In this study, we describe in vitro properties of A-867744, a novel and selective α7 PAM. Similar to PNU-120596 and TQS (Grønlien et al., 2007), A-867744 potentiated ACh-evoked α7 currents in a manner characteristic of type II PAMs (i.e., increasing peak current and strongly affecting the apparent current decay). Although at highest concentrations tested, A-867744, just like TQS and PNU-120596, produced essentially nondecaying agonist-evoked current profile, at lower concentrations a qualitative difference was noted. In contrast to TQS and PNU-120596, A-867744 did not evoke a distinct secondary component, suggesting existence of additional types of PAM profiles. The potentiating effect of A-867744 was observed at recombinant human or rat α7 expressed in oocytes and native rat α7 receptors in the hippocampus (CA1 interneurons) and dentate gyrus. In the presence of A-867744, the responses to ACh were potentiated by increases in the Hill slope, potency, and maximal efficacy. Furthermore, A-867744 accelerated the time course of α7 current recovery from desensitization in hippocampus slices, a finding not previously shown for any α7 PAM. A-867744 failed to enhance responses at other nicotinic or 5-HT3A subunits examined, and it did not increase responses mediated by chimeric human α7/5-HT3 receptors, composed of the N terminus and M2-3 loop sequence of human α7 and the remainder of human 5-HT3A. A-867744, similar to PNU-120596, TQS, and NS1738, did not displace the binding of [3H]MLA to α7 receptor consistent with the interaction of these compounds with an allosteric site. However, A-867744, unlike these three PAMs, displaced the binding [3H]A-585539 to α7 receptors. This displacement did not occur in the α7/5-HT3 chimera, which retains the ligand binding N-terminal domain of the α7 receptor, and was activated by α7 agonists. Our data show that A-867744 is a novel α7 PAM exhibiting a unique pharmacological interaction with the α7 nAChR.
In addition to A-867744, several other compounds have been described to function as α7 nAChR PAMs: 5-HI, ivermectin, genistein, NS1738, CCMI, galantamine, LY-2087101, PNU-120596, TQS, and SB-206553 (for review, see Lightfoot et al., 2008; Bertrand and Gopalakrishnan, 2007). Based on the manner by which α7 current potentiation occurs, at least two types of PAMs are distinguished. Type I PAMs exhibit a predominant effect on peak current amplitude with little effect on current decay, exemplified by NS1738, 5-HI, and genistein (Zwart et al., 2002; Grønlien et al., 2007; Timmermann et al., 2007). A-867744 exhibits a different profile similar to that of TQS and PNU-120596 (Hurst et al., 2005; Grønlien et al., 2007). These compounds, classified as type II PAMs, also increase the peak current amplitude and have a strong effect on the current decay profile. At highest concentrations tested for type II PAMs, the α7 agonist-evoked currents are essentially nondecaying. The effects at lower concentrations tested revealed an important difference between A-867744 and the other type II PAMs under the conditions used in this study (Fig. 1) (Grønlien et al., 2007). Unlike TQS or PNU-120596, A-867744 was not able to activate a distinct secondary component. This suggests that type II PAMs may be further divided. Another property of type II PAMs, including for A-867744 (Fig. 1e), is that they are able to reactivate desensitized α7 nAChRs, unlike type I PAMs (Hurst et al., 2005; Grønlien et al., 2007). We further provide evidence that the reactivation depends on α7 nAChR activity because MLA was able to attenuate the responses to A-867744 in the presence of an α7 agonist, NS6784 (Fig. 3).
A-867744 similar to TQS, PNU-120596, and NS1738 did not displace the specific binding of [3H]MLA to native α7 nAChRs or [3H]cytisine to native α4β2* nAChRs (Timmermann et al., 2007; this study). Because MLA binds to orthosteric site on the α7 nAChR, these results support the binding of these compounds to an allosteric site (or multiple allosteric sites). Recently, a novel α7 radioligand, [3H]A-585539, was described, showing a Kd value of ∼0.065 nM at α7 nAChRs, and ∼0.8 nM, to a chimeric α7/5-HT3 receptor composed of N terminus of human α7 and the rest of human 5-HT3A (Anderson et al., 2008; Bertrand et al., 2008). In the present study, we report a Kd value of 0.15 nM in chimera containing an additional α7 encoded M2-3 loop, indicating an ∼5-fold increase in affinity. In addition, A-867744, but not the NS1738, TQS, or PNU-120596, displaced the binding of [3H]A-585539 in the brain. In contrast, all four compounds were ineffective at displacing [3H]A-585539 or [3H]MLA in the chimera (Fig. 6). Although the mechanism responsible for the differential profile of A-867744 versus other PAMs remains to be established, we hypothesize that the same allosteric site interaction is involved in the displacement of [3H]A-585539 binding and positive modulation at the α7 nAChR. An alternative explanation might be that there are at least two distinct sites: one responsible for displacement of [3H]A-585539 and the other for its PAM activity both involving domains of the α7 nAChR distinct from the N terminus and M2-3 loop.
A-867744 was effective in comparably potentiating recombinant human and rat α7 nAChRs and also in enhancing agonist evoked native α7 currents in rat hippocampus CA1 interneurons. This effect on CA1 interneurons is similar to that reported previously for PNU-120596 (Hurst et al., 2005). We further quantify that in the presence of A-867744, α7 currents in the CA1 hippocampus were enhanced at least 2-fold in amplitude, total charge transfer, half-width, and current decay τ (Table 2). We also show that A-867744 was able to reduce the time course of recovery of α7 current from desensitization. Such effect, to our knowledge, has not yet been described for any type II PAM. In the case of type I PAMs, there is a report showing that 5-HI slowed the time course of recovery of α7 currents (Zwart et al., 2002), an opposite effect to that observed in this study for A-867744. Whether this difference is specific for these two PAMs or constitutes a fundamental type I versus II distinction remains to be elucidated. It should also be noted that the process of α7 current recovery from desensitization is complex and involves dissociation of ligand from multiple sites and conformational state transitions (Palma et al., 1996; van Hooft and Vijverberg, 1996). Any given PAM may, hence, affect the on and off rates of the agonist, in addition to its on- and off-rate kinetics and the conformational transitions in the basal-open-desensitized triad, including reversal of desensitized receptors favoring the open active state.
Both types of α7 PAMs have been reported to improve cognitive function or sensory gating deficits (Hurst et al., 2005; Ng et al., 2007; Timmermann et al., 2007). Likewise, in vivo studies with A-867744 in the DBA/2 mouse model showed significant improvement in sensory gating deficits (Faghih et al., 2009). Although molecular and cellular mechanisms responsible for these in vivo observations remain to be elucidated, it is likely that effects on synaptic transmission play a role as shown by the responses of 5-HI and PNU-120596 in rat cerebellum and hippocampus (Frazier et al., 2003; Hurst et al., 2005; Thinschmidt et al., 2008). Similarly in this study, A-867744 enhanced choline evoked spontaneous GABAergic IPSC activity in dentate gyrus.
Both α7 nAChRs and 5-HT3 receptors share substantial homology, which has been used experimentally by swapping homologous domains generating functional chimeric receptors. The earliest reported chimera contained the N-terminal region of chick α7 fused to mouse 5-HT3 (Eiselé et al., 1993). Other reported chimeras involved the N-terminal region of human α7 and the rest of sequence encoded by mouse 5-HT3 (Craig et al., 2004), the N-terminal region of rat α7 and the remainder being mouse 5-HT3 (Young et al., 2008), and the N-terminal sequence of human α7 and the remainder of human 5-HT3A (Bertrand et al., 2008). Additional human α7/human 5-HT3A chimeras were also described previously (Bertrand et al., 2008). One of which contained the N terminus and M2-3 extracellular loop of human α7 sequence and the rest human 5-HT3A. In this study, we show that this chimera is also functional when stably expressed in HEK-293 cells and assessed using patch clamp and membrane potential imaging (Fig. 5). It is important to note that A-867744 failed to potentiate agonist-evoked chimeric responses whether obtained by submaximal (Fig. 5) or supramaximal (chimeric current expressed in X. laevis oocytes and evoked by 100 μM ACh; data not shown) concentrations, indicating that its site of interaction probably involves regions beyond the N-terminal domain and M2-3 extracellular loop. A recent report has provided evidence that PNU-120596 and LY-2087101, type II and I PAMs, respectively, interact within the transmembrane domain at two critical amino acids (Ala225 and Met253) using single-site mutagenesis of chimeric receptors (Young et al., 2008). Taken together, these studies show that there are multiple binding sites for α7 nAChR allosteric potentiators.
In summary, this study identifies a novel and selective type II α7 nAChR PAM, A-867744, active at both recombinant and native α7 nAChRs that exhibits unique pharmacological interaction with the α7 nAChR. Availability of tools such as A-867744 further expands opportunities to elucidate the underlying mechanisms involved in allosteric modulation of the α7 nAChR.
This work was supported by Abbott. All authors are employees of Abbott.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: nAChR, neuronal acetylcholine receptor; 5-HT, 5-hydroxytryptamine; PAM, positive allosteric modulator; SB-206553, 3,5-dihydro-5-methyl-N-3-pyridinylbenzo[1,2-b:4,5-b′]di pyrrole-1(2H)-carboxamide; PNU-120596, 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea; TQS, 4-naphthalen-1-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonic acid amide; CCMI (XY4083), N-(4-chlorophenyl)-[[(4-chlorophenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide; 5-HI, 5-hydroxyindole; A-867744, 4-(5-(4-chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)-benzenesulfonamide; MLA, methyllycaconitine; PNU-282987, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride; A-585539, (1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane; NS6784, 4-(5-phenyl-[1,3,4]oxadiazol-2-yl)-1,4-diaza-bicyclo-[3.2.2]nonane; NS1738, 1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)urea; TTX, tetrodotoxin; APV, 2-amino-5-phosphonovalerate; CNQX, 6-cyano-2,3-dihydroxy-7-nitroquinoxaline; FMP, fast membrane potential; HEK, human embryonic kidney; FLIPR, fluorometric imaging plate reader; LY-2087101, [2-(4-fluoro-phenylamino)-4-methyl-thiazol-5-yl]-thiopen-3-yl-methanone; ACSF, artificial cerebrospinal fluid; IPSC, inhibitory postsynaptic current; P, puff application; Hpot, holding potential.
- Received February 11, 2009.
- Accepted April 21, 2009.
- The American Society for Pharmacology and Experimental Therapeutics