Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Nov 1.
Published in final edited form as: J Pharmacol Exp Ther. 2008 Jul 29;327(2):529–537. doi: 10.1124/jpet.108.142943

αConotoxin ArIB[V11L,V16D] is a potent and selective antagonist at rat and human native α7 nicotinic acetylcholine receptors

Neal Innocent 1, Phil D Livingstone 1, Arik Hone 1, Atsuko Kimura 1, Tracey Young 1, Paul Whiteaker 1, J Michael McIntosh 1, Susan Wonnacott 1
PMCID: PMC2596936  NIHMSID: NIHMS63464  PMID: 18664588

Abstract

A recently developed α-conotoxin, α-CtxArIB[V11L,V16D] is a potent and selective competitive antagonist at rat recombinant α7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. α7 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. Here we evaluate this toxin at rat and human native nAChRs. Functional α7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 in rat PC12 cells and human SHSY5Y cells loaded with calcium indicators. α-CtxArIB[V11L,V16D] specifically inhibited α7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli (5-iodo-A-85380, nicotine or KCl) that did not activate α7 nAChRs were unaffected. Human α7 nAChRs were also sensitive to α-CtxArIB[V11L,V16D]: ACh-evoked currents in X. laevis oocytes expressing human α7 nAChRs were inhibited by α-CtxArIB[V11L,V16D] (IC50 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the timecourse of recovery from blockade of rat α7 nAChRs in PC12 cells. α-CtxArIB[V11L,V16D] inhibited human native α7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 plus PNU-120596. Rat brain α7 nAChRs contribute to dopamine release from striatal minces: α-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 α-CtxArIB[V11L,V16D] selectively inhibits human and rat native α7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating α7 nAChR functions.

Introduction

The α7 subtype of nicotinic acetylcholine receptor (nAChR) is currently attracting considerable interest for its roles in brain function, including synaptic plasticity (McKay et al., 2007) and attentional aspects of cognitive performance (Levin et al., 2006). Consequently, α7 nAChRs are therapeutic targets for cognitive impairments in Alzheimer’s disease, schizophrenia and attention deficit hyperactivity disorder (Mazurov et al., 2006; Chiamulera and Fumagalli, 2007; Wilens and Decker, 2007). α7 nAChRs also have therapeutic potential in several other processes, including pain, inflammation and wound healing (Decker and Meyer, 1999; de Jonge and Ulloa, 2007). Selective antagonists are essential research tools for elucidating and clarifying the molecular and cellular mechanisms that underpin these effects.

Research into α7 nAChRs has profited from the availability of two useful antagonists: αbungarotoxin (αBgt) and methyllycaconitine (MLA). αBgt, from the venom of the Elapidae snake Bungarus multicinctus, is a large polypeptide with slow binding kinetics. This confers high affinity binding with very slow dissociation, an advantage for some studies. It can also be a limitation in functional assays as increased incubation times and/or concentrations are required to achieve blockade. Moreover the slowly reversible block limits recovery in experiments where repeated agonist application is desirable, and the large size of the toxin can compromise its access to α7 nAChRs in intact preparations.

The norditerpenoid MLA from Delphinium and Consolida sp. is also a competitive antagonist of α7 nAChRs. It binds to α7 nAChRs with nanomolar affinity (Ki ∼ 1 nM; Davies et al., 1999). In addition, it exhibits moderately high affinity for α6β2-containing nAChRs (Ki 33 nM; Mogg et al., 2002), which compromises its utility for identifying α7 nAChRs in areas where the α6 subunit is expressed, notably catecholaminergic and visual pathways. Furthermore, both αBgt and MLA interact with high affinity with nAChRs containing α9 and α10 subunits (Elgoyhen et al., 2001; Baker et al., 2004). The potential co-localisation of α7, α9 and α10 subunits in sympathetic neurones (Lips et al., 2006) and non-neuronal cells (Bschleipfer et al., 2007) demands more discriminating antagonists.

The α-conotoxins, small peptides from the venom of Conus sp., can exhibit exquisite specificity for nAChR subtypes (McIntosh et al., 1999; Nicke et al., 2004). Recently, a naturally occurring αconotoxin from C. arenatus was modified by changing two amino acid residues to generate a novel toxin α-CtxArIB[V11L,V16D] (Fig. 1A; Whiteaker et al., 2007). This toxin displayed high potency and specificity for discriminating α7 nAChRs (Ki 7 nM) from all other nAChR subtypes tested in binding assays. Functionally, α-CtxArIB[V11L,V16D] specifically blocked currents evoked by stimulation of rat homomeric α7 nAChRs expressed in Xenopus oocytes, whereas it was without effect on heteromeric nAChRs (including α9α10 nAChR) in the same expression system (Whiteaker et al., 2007). However, the subunit composition of native nAChRs can be more diverse and more complex than that of recombinant receptors. Here we have assessed the ability of α-CtxArIB[V11L,V16D] to antagonise responses to native nAChRs.

Figure 1.

Figure 1

Open in a new tab

(A) Peptide sequence of α-CtxArIB[V11L,V16D]. Bars indicate the disulphide bond pattern and the mutated amino acids are underlined. (B) Inhibition of 125I-αBgt binding to rat brain membranes by α-CtxArIB[V11L,V16D] (solid line) or nicotine (dashed line). Data points were fitted to the Hill equation, giving Ki values of 4.0 ± 0.6 nM and 2.3 ± 0.6 μM, respectively. Data represent mean ± SEM, n = 3 (C) α-CtxArIB[V11L,V16D] has no effect on [3H]epibatidine binding to rat brain membranes (solid line), whereas nicotine displaced [3H]epibatidine binding, Ki value 22.5 ± 16.8 nM (dashed line). Data represent mean ± range, n = 2.

Clinical studies demand investigation of human tissues. The exquisite specificity of α-conotoxins for particular nAChR subtypes (Nicke et al., 2004), coupled with subtle species differences that can affect pharmacological selectivity (Young et al., 2007), requires caution when extrapolating from rodent to human tissues. Therefore we have exploited both PC12 and SH-SY5Y cell lines that are representative of rat and human peripheral autonomic neurones, respectively. Both cell lines express α3, α5, α7, β2 and β4 nAChR subunits (Henderson et al. 1994; Blumenthal et al. 1997; Lukas et al., 1993; Peng et al., 1994; Wang et al., 1996) and have functional α7 and non-α7 nAChRs that can be discriminated using subtype-selective ligands (Dickinson et al., 2007). Additional nAChR subtypes are expressed in the brain, and their heterogeneity has been particularly well defined with respect to nAChR-evoked [3H]dopamine release from rodent striatal preparations. Several β2-containing heteromeric nAChRs (α4β2, α4α5β2, α4α6β2β3, α6β2β3, α6β2) reside on dopamine terminals (Grady et al., 2007). In addition, α7 nAChRs assigned to neighbouring glutamate afferents can also modulate striatal [3H]dopamine release (Kaiser and Wonnacott, 2000; Barik and Wonnacott, 2006). We show that in rat striatal minces, as well as in the rat and human cell lines, α-CtxArIB[V11L,V16D] selectively inhibited responses attributable to α7 nAChRs while having no effect on non-α7 nAChR responses. These results establish the utility of this new antagonist for interrogating the actions of rat and human α7 nAChRs in vitro.

Materials and Methods

Materials

[7,8-3H]Dopamine (43 Ci/mmol) and [125I]-αbungarotoxin (263 Ci/mmol) were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). The PC12 cell line was a gift from D. Berg (University of California, San Diego, USA). SH-SY5Y cells were obtained from ECACC, Sailsbury, UK. 5-Iodo-A85380 dihydrochloride (5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride. 5-I-A85380) and PNU-120596 (1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea) were purchased from Tocris Cookson (Bristol, Avon, UK). α-Bungarotoxin (αBgt), fluo-3 AM and pluronic F127 were purchased from Invitrogen (Poortgebouw, Netherlands). (-)-Nicotine hydrogen tartrate, pargyline hydrochloride, choline tartrate, ascorbic acid, nomifensine maleate, mecamylamine, RPMI 1640 medium, horse serum, foetal bovine serum, L-glutamine, penicillin and streptomycin were obtained from Sigma-Aldrich Co. (Poole, Dorset, UK). AR-R17779 ((2)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin]-29-one) was provided by GlaxoSmithKline, (Harlow, UK). Optiplates and Optiphase Supermix were obtained from Perkin Elmer (N.V./S.A., Zaventem, Belgium). Other standard chemicals of analytical grade were attained from Sigma-Aldrich Co. Male Sprague-Dawley rats (approximately 250g) were from Charles River, UK.

Nicotine tartrate, choline and KCl solutions were prepared freshly for each experiment by dissolving in assay buffer and correcting the pH to 7.4 if necessary. Other drugs were diluted from stocks made up in water, with the exception of PNU-120596 (100 mM stock in dimethylsulfoxide).

Radioligand binding

Competition binding experiments for 125I-αBgt and [3H]-epibatidine binding sites were carried out on rat brain P2 membranes as previously described (Davies et al.., 1999; Whiteaker et al., 2000). For 125I-αBgt, membranes (250 μg) were incubated in a total volume of 1ml in 10 mM phosphate buffer, pH 7.4, containing 0.1 % bovine serum albumin (BSA) and 1 nM 125I-αBgt in the presence and absence of increasing concentrations of α-CtxArIB[V11L,V16D] or (-)-nicotine for 4 h at 37°C, followed by chilling to 4°C. Samples were filtered through a double thickness of Gelman A/E filters, pre-soaked overnight in 0.3 % polyethyleneimine (bottom filter) or 4 % milk powder (top filter), using a Brandel Cell Harvester. For [3H]-epibatidine, membranes (250 μg) were incubated in a total volume of 2ml in 10 mM phosphate buffer, containing 0.2 nM [3H]-epibatidine in the presence and absence of increasing concentrations of α-CtxArIB[V11L,V16D] or (-)-nicotine for 2 h at room temperature. Samples were filtered through a double thickness of Gelman A/E filters, pre-soaked overnight in 0.3 % polyethyleneimine, using a Brandel Cell Harvester. Nonspecific binding was determined in the presence of 1 mM nicotine.

Filters were subsequently washed five times using cold PBS and counted for radioactivity in a Packard 1600 Tricarb scintillation counter (Packard Instrument Company, Meriden, CT, USA).

Cell culture

PC12 cells were cultured as described by Dickinson et al. (2007). Cells were grown in a humidified incubator under 7% CO2 at 37°C in RPMI medium supplemented with 5% foetal bovine serum, 10% heat inactivated donor horse serum, 2 mM L-glutamine and 50 U/ml penicillin/streptomycin. Cells were plated at a density of 2.5 × 104 cells/cm2 onto 25 mm glass coverslips in 6 well plates or into 96 well plates, both coated with poly-D-lysine (5 μg/ml). Assays were conducted 24 h later with confluent cultures.

Human neuroblastoma SH-SY5Y cells were cultured as described previously (Ridley et al., 2001). Cultures were maintained in 175 cm2 tissue culture flasks in DMEM:F12, supplemented with 15 % foetal bovine serum, 2 mM L-glutamine, 1 % non-essential amino acids, 190 U/ml penicillin and 0.2 mg/ml streptomycin, until confluent. Cells were plated (1:5 dilution) into 96 well plates and experiments performed 72 h later with confluent cultures.

Calcium fluorimetry (1) cell populations

Increases in intracellular Ca2+ in PC12 and SH-SY5Y cells were monitored by measuring changes in fluorescence in cells loaded with fluo-3, as previously described (Dickinson et al., 2007). In brief, cells cultured in 96 well plates were washed twice with Tyrode’s salt solution (TSS, in mM: NaCl, 137.0; KCl, 2.7; MgCl2, 1.0; CaCl2, 2.5; NaH2PO4, 0.2; NaHCO3, 12.0; glucose, 5.5; pH 7.4; supplemented with 0.1% bovine serum albumin (BSA) to prevent α-CtxArIB[V11L,V16D] binding to plastic or glass) and incubated with fluo-3 AM (10 μM) and 0.02% pluronic F127 for 1 h at room temperature (22°C) in the dark. After washing the cells twice with TSS, cells were preincubated with 80 μl TSS, with or without antagonist (10 min for αCtxArIB[V11L,V16D], mecamylamine or DHβE; 20 min for αBgt). Where used, PNU-120596 was added I min prior to the end of preincubation. Basal fluorescence (excitation 485 nm, emission 538 nm) was recorded for 5 s in a Fluoroskan Ascent fluorescent plate reader (Labsystems, Helsinki, Finland). Then nAChR agonist, KCl or vehicle (20 μl) was added and the change in fluorescence was recorded for a further 20 s. Under all conditions examined, increases in fluorescence reached a maximum level within 5 s and this response was sustained over the remainder of the time course of the experiment. In order to normalise fluo-3 signals, the maximum and minimum fluorescence from each well was determined by addition of 0.3% Triton-X100 (Fmax) followed by 100 mM MnCl2 (Fmin). Drug-evoked responses were expressed as a percentage of the corresponding (Fmax-Fmin) value. None of the drugs or vehicles used significantly altered the basal level of fluorescence.

Calcium fluorimetry (2) timecourse experiments

Changes in intracellular Ca2+ in individual PC12 cells loaded with fura-2 were monitored using dynamic video imaging (Concord System, Perkin Elmer, UK). PC12 cells cultured on coverslips were washed twice with Ca2+ buffer (in mM NaCl, 140.0; KCl, 5.0; MgCl2, 1.0; CaCl2, 1.8; glucose, 10.0; HEPES, 5.0; pH 7.4; supplemented with 0.1% BSA) and incubated with fura-2 AM (5 μM) and 0.02% pluronic F127 for 1 h at room temperature (22°C) in the dark. Coverslips were placed in a temperature-controlled microscope chamber maintained at 37 °C (Harvard Apparatus, MA) and continuously perfused with buffer (5 ml/min). Agonist with or without α-CtxArIB[V11L,V16D] was applied via the perfusion system for 1 min, followed by washout before re-application of agonist alone. Fura-2 was excited at 340 and 380 nm using a SpectroMaster I (Perkin Elmer, UK) and emssions at 510 nm were detected with an intensified Ultrapix PDCI low light level CCD camera (Perkin Elmer, UK). Data were analysed using Ultraview software (Perkin Elmer, UK) and expressed as a ratio of F340:F380. Each cell that responded to agonist was analysed separately and these data were pooled for each experiment.

cRNA preparation and injection

Human α7 nAChR subunit cDNA was sub-cloned into the RNA expression vector pBluescript II SK- (Stratagene, La Jolla, CA) and the resulting construct (from J. Garrett, Cognetix Inc., Salt Lake City, UT) was used to make cRNA. Capped cRNA was made using the mMessage mMachine in vitro transcription kit (Ambion, Austin, TX) following linearization of the plasmid with NotI and transcription under the control of a T7 promotor. The cRNA was purified using the Qiagen RNeasy kit (Qiagen, Valencia, CA). The concentration of cRNA was determined by the absorbance at 260 nm. Fifty nl (608 ng/μl) of cRNA was injected into each Xenopus oocyte with a Drummond microdispenser (Drummond Scientific, Broomall, PA), as described previously (Cartier et al., 1996), and incubated at 17 °C. Oocytes were injected within one day of harvesting and recordings were made 3-6 days post-injection.

Voltage-clamp Recording

Oocytes were voltage-clamped and exposed to ACh and peptide as described previously (Cartier et al., 1996). Briefly, the oocyte chamber consisting of a cylindrical well (∼30 μl in volume) was gravity perfused at a rate of ∼2 ml/min with ND-96 buffer (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES, pH 7.5) containing 0.1 mg/ml BSA. Oocytes were subjected once a minute to a 1 sec pulse of 200 μM ACh. Once a stable baseline was achieved, either ND-96 alone or ND-96 containing varying concentrations of α-CtxArIB[V11L;V16D] was perfusion-applied during which 1-sec pulses of 200 μM ACh were applied every minute until a constant level of block was achieved. For experiments involving PNU-120596, the ACh concentration was reduced to 20 μM and PNU-120596 was added to the ND-96 at a final concentration of 10 μM.

Data Analysis

For the baseline response, at least 3 ACh-induced responses were averaged. To determine the percent block induced by toxin, after a steady-state block had been achieved, ACh-induced responses were averaged and the value divided by the pre-toxin baseline value to yield a % response. The concentration-response data were fit to the equation, Y = 100/(1 + 10^((LogEC50 - Log[Toxin]) × nH)), where nH is the Hill coefficient, by non-linear regression analysis using GraphPad Prism (GraphPad Software, San Diego, CA). Each data point represents the mean ± SEM from at least 4 oocytes.

[3H]Dopamine Release

A 96 well filter plate assay was used to monitor [3H]dopamine release from striatal minces (Puttfarken et al., 2000; Barik & Wonnacott, 2006). For each experiment, 2 rats were killed by cervical dislocation, brains were rapidly removed and striata dissected and transferred to ice cold Krebs buffer (KB, in mM: NaCl 118.0, KCl 2.4, CaCl2 2.4, KH2PO4 1.2, MgSO4.7H2O 1.2, NaHCO3 25, D-glucose 10, ascorbic acid 1, pargyline 0.01, gassed with 95 % O2 and 5 % CO2 for at least 1 h at 37 °C; pH 7.4). Tissue was chopped using a McIlwain tissue chopper to give 150 μm prisms. Following 2 washes with warm KB, striatal minces were incubated for 30 min with 50 nM [3H]dopamine in 5 ml KB at 37°C. After 5 washes in KB containing 0.5 μM nomifensine, minces (100 μl / well) were loaded into a 96 well filter plate (model MABVN1250, Millipore, Hertfordshire, UK) and preincubated for 10 min with buffer (70 μl) in the presence or absence of antagonist or modulator. Buffer was removed by filtration and replaced with fresh buffer (70 μl) containing agonist and/or antagonist. After a further 5 min at 37°C, buffer containing released [3H]dopamine was collected by filtration into a 96-well Packard Optiplate™ (Perkin Elmer, Belgium). Filters were removed to a separate Optiplate containing 70 μl water/well. Radioactivity was determined using a Microbeta liquid scintillation counter (Wallac 1450 Microbeta Trilux, Perkin Elmer, Finland), counting efficiency 30%. The amount of [3H]dopamine released was expressed as a percent of total radioactivity present in the slices prior to stimulation (i.e. tritium remaining in the tissue + amount of tritium released). Each condition was examined in 6 replicates and repeated in at least 3 independent experiments.

Statistics

Statistical significance (P < 0.05) was determined using student’s t-test, carried out using Sigma Plot V8.0.

Results

α-CtxArIB[V11L,V16D] (Fig. 1A) potently displaced 125I-α-bungarotoxin binding to rat P2 membranes with a Ki value of 4.0 ± 0.6 nM (Fig 1B), confirming that α-CtxArIB[V11L,V16D] interacts competitively at α7 nAChRs, as previously described (Whiteaker et al., 2007). α-CtxArIB[V11L,V16D] did not compete for [3H]-epibatidine (0.2 nM) binding to rat brain membranes (Fig 1C). At this concentration, [3H]-epibatidine will not label α7 nAChRs (Ki ∼ 0.2 μM, Davies et al., 1999, see Marks et al, 2006). Therefore this result indicates that over the concentration range 0.1 nM - 1 μM α-CtxArIB[V11L,V16D] does not interact with non-α7 nAChRs. α-CtxArIB[V11L,V16D] was then tested for functional potency at native nAChRs.

Effects of α-CtxArIB[V11L,V16D] on nAChR-mediated Ca2+ responses in PC12 cells

PC12 cells express rat α7 and non-α7 nAChRs (Blumenthal et al. 1997) and we have recently characterised the differential coupling of these nAChR subtypes to increases in intracellular Ca2+ (Dickinson et al. 2007). Here, we first used choline to increase Ca2+ by selectively activating α7 nAChRs. Choline (1, 3 mM) elicited very small increases in fluorescence in PC12 cells loaded with fluo-3 AM but these responses were substantially amplified in the presence of 10 μM PNU-120596, an α7-selective positive allosteric modulator (Fig 2A,B; Hurst et al., 2005). PNU-120596 alone produced no response (data not shown). αBgt (100 nM; 20 min pre-incubation) reduced responses to 1 mM and 3 mM choline plus PNU-120596 by 90.9 ± 0.9 % and 80.6 ± 1.8 %, respectively (Fig 2B).

Figure 2.

Figure 2

Open in a new tab

Effect of α-CtxArIB[V11L,V16D] (α-Ctx) on Ca2+ responses in PC12 cells. PC12 cells loaded with fluo-3 AM were preincubated with α-CtxArIB[V11L,V16D] for 10 min, with or without PNU-120596 (PNU) for the last 60 s, and stimulated with agonist or KCl as described in Materials and Methods. The change in fluorescence was measured over 20 s (A) Representative traces of change in fluore scence in response to 1 mM choline, in the absence (dotted line) or presence of 10 μM PNU-120596 (PNU) (solid line) or with 10 μM PNU-120596 and 100 nM α-CtxArIB[V11L,V16D] (α-Ctx) (dashed line) (B) Increases in fluorescence evoked by 1 and 3 mM choline, with or without 10 μM PNU-120596, were measured in the presence and absence of 100 nM αBgt (mean ± range, n = 2) or 100 nM α-CtxArIB[V11L,V16D] (mean ± SEM, n = 4; ** p < 0.01, student’s t-test) (C) The concentration-dependence of inhibition by α-CtxArIB[V11L,V16D] of responses to 1 mM choline plus 10 μM PNU-120596. Data points were fitted to the Hill equation, giving an IC50 value of 88.0 ± 17.4 nM (mean ± SEM, n = 4) (D) Effect of 300 nM α-CtxArIB[V11L,V16D] (hatched bars) on increases in fluorescence in response to 100 μM nicotine (with or without 10 M PNU-120596), 30 μM 5-I-A85380 (5-I-A) or 60 mM KCl. PNU-120596 significantly increased responses to nicotine in the absence (but not in the presence) of 300 nM α-CtxArIB[V11L,V16D] (mean ± SEM, n = 4; * p < 0.05, student’s t-test). (E) Timecourse of recovery from inhibition by α-CtxArIB[V11L;V16D]. PC12 cells were loaded with fura-2 AM and perfused with buffer as described in Materials and Methods. Fluorescence changes in response to one minute applications of 3 mM choline and 10 μM PNU-120596 (PNU), in the presence and absence of 300 nM α-CtxArIB[V11L,V16D] (α-Ctx), were monitored. A representative trace from one experiment in which responses from 13 cells that responded to agonist stimulation have been combined.

In initial experiments, cells were pre-incubated with 100 nM α-CtxArIB[V11L,V16D] for 10 min prior to stimulation with choline (1 and 3 mM) and 10 μM PNU-120596. This concentration of α-CtxArIB[V11L,V16D] significantly inhibited responses, by 62.0 ± 5.4 % and 49.9 ± 6.2 %, respectively (Fig. 2B). α-CtxArIB[V11L,V16D] was then tested over the concentration range 10 - 300 nM against 1 mM choline plus PNU-120596. α-CtxArIB[V11L,V16D] blocked α7 nAChR-mediated responses in a concentration-dependent manner with an IC50 of 88.0 ± 17.4 nM (Fig. 2C). Maximum inhibition of 83.6 % was observed with 300 nM α-CtxArIB[V11L,V16D]. Higher concentrations were not tested in order to conserve the toxin.

To assess the specificity of α-CtxArIB[V11L,V16D] for α7 nAChR, its effects on responses induced by a non-selective nAChR agonist (nicotine), an agonist selective for β2-containing nAChRs (5-I-A85380) and a general depolarising agent (KCl) were examined (Dickinson et al., 2007). Nicotine (100 μM), 5-I-A85380 (30 μM) and KCl (60 mM) elicited increases in fluorescence of 26.7 ± 2.1 %, 14.8 ± 1.7 % and 44.2 ± 3.7 %, respectively (Fig 2D, mean ± SEM, n = 4). α-CtxArIB[V11L,V16D], tested at the highest concentration used previously (300 nM; Fig. 2C), failed to reduce the responses to any of these stimuli. To determine if 100 μM nicotine activated α7 nAChRs in addition to non-α7 nAChRs, it was applied in the presence of PNU-120596 (10 μM). Under this condition responses were modestly enhanced by 53.6% (from 27 % to 41 % of the maximum response, in the absence and presence of PNU-120596 respectively). This is consistent with the response to 100 μM nicotine including a small α7 nAChR-mediated component. In the presence of α-CtxArIB[V11L,V16D], the PNU-120596-enhanced response was not observed (Fig. 2D).

The timecourse of recovery from blockade by 300 nM α-CtxArIB[V11L,V16D] was assessed in PC12 cells loaded with fura2 AM and monitored using dynamic video imaging. The response to 3 mM choline plus 10 μM PNU-120596 (60 s) was blocked by 300 nM α-CtxArIB[V11L,V16D] by 87.1 ± 1.6 % (mean ± S.E.M. from 34 agonist-responsive cells [40.5 % of total number of cells monitored] in 3 separate experiments). Following removal of the toxin from the perfusing buffer, responses to choline plus PNU-120596 slowly recovered, with complete recovery after 15 min washout (Fig. 2E).

Blockade of human α7 nAChR-mediated responses by α-CtxArIB[V11L,V16D]

To determine if α-CtxArIB[V11L,V16D] is similarly effective at human α7 nAChRs, the toxin was examined for blockade of human α7 nAChRs heterologously expressed in X. laevis oocytes. Responses to 200 μM ACh by oocytes transfected with human α7 nAChR were blocked by α-CtxArIB[V11L,V16D] with an IC50 value of 3.4 ± 0.037 (2.9-4.11) nM and a Hill slope of 1.20 ± 0.12 (1.5-1.0) (Fig. 3A). A maximum block of 100 % was obtained with 100 nM toxin. Repeating this experiment with 20 μM ACh plus 10 μM PNU-120596 resulted in a slight rightward shift of the concentration response curve for inhibition by α-CtxArIB[V11L,V16D]; the IC50 value was increased 2-fold (6.7 nM; Fig. 3A). This indicates that amplification of α7 nAChR responses by PNU-120596 does not significantly perturb the sensitivity to toxin. Kinetic analysis shows that α-CtxArIB[V11L,V16D] blocks α7 nAChR currents evoked by 200 μM ACh within 60 s of exposure, with responses beginning to recover 180 s after removal of the toxin (Fig. 3B).

Figure 3.

Figure 3

Open in a new tab

Effect of α-CtxArIB[V11L;V16D] on human α7 nAChRs heterologously expressed in X. laevis oocytes. (A) Concentration-response analysis of peptide block of ACh-induced currents was performed as described in the Materials and Methods. α-CtxArIB[V11L;V16D] blocked human α7 nAChRs activated by 200 μM ACh (squares) with an IC50 value of 3.4 ± 0.04 (2.9-4.1) nM. The Hill slope was 1.2 ± 0.12 (1.5-1.0). Values are the mean ± SEM from an n of 4-5 for each concentration. Activation of currents by 20 μM ACh plus 10 μM PNU-120596 yielded an IC50 value of 6.7 ± .05 (5.3-8.6) nM (diamonds). Values are the mean ± SEM from an n of 3-4 for each concentration. The Hill slope was 1.1 ± 0.13 (1.4-0.8). 95% confidence interval is given in parentheses. (B) Kinetics of block and unblock. A representative trace recording of current amplitudes induced by 200 μM ACh applied to human α7 nAChRs heterologously expressed in X. laevis oocytes before, during, and after perfusion with 100 nM α-CtxArIB[V11L;V16D]. c; control, prior to toxin.

SH-SY5Y cells resemble human foetal sympathetic neurones grown in primary cultures and express a similar range of nAChR subunits to the PC12 cell line (Lukas et al.,1993; Peng et al.,1994). Changes in intracellular Ca2+ in SH-SY5Y cells loaded with fluo-3 were determined to assess the effects of α-CtxArIB[V11L,V16D] on native human α7 nAChRs. In agreement with the data from PC12 cells (Fig. 2), 3 mM choline alone was without effect but in the presence of 10 μM PNU-120596 it evoked an increase in intracellular Ca2+ in SH-SY5Y cells that was inhibited by αBgt by 93.4 ± 1.0 %, whereas DHβE had no significant effect (Fig. 4A). α-CtxArIB[V11L,V16D] produced a concentration-dependent inhibition of responses to choline plus PNU-120596 with an IC50 value of 74.0 ± 1.8 nM. Another α7 nAChR-selective agonist, AR-R17779 (Jensen et al., 2005; Fig. 4B) was examined for comparison with choline. This agonist was also ineffective in the absence of PNU-120596; responses in the presence of the allosteric modulator were blocked by αBgt by 95.4 ± 1.8 % but not by DHβE. The concentration-dependence of inhibition by α-CtxArIB[V11L,V16D] resulted in an IC50 value of 51 ± 1.0 nM; maximum inhibition by 1 μM α-CtxArIB[V11L,V16D] was 95.9 %.

Figure 4.

Figure 4

Open in a new tab

Effect of α-CtxArIB[V11L,V16D] on native human α7 nAChRs in SH-SY5Y cells. SH-SY5Y cells were loaded with fluo-3 AM and preincubated with inhibitors for 10 min and / or PNU-120596 for 1 min before stimulation with choline (A) or AR-R17779 (B), as described in the Materials and Methods. Increases in fluorescence evoked by agonist were measured in the presence or absence of 100 nM αBgt, 1 μM DHβE or increasing concentrations of α-CtxArIB[V11L,V16D]. Insets: Concentration response curve for inhibition of responses to agonist plus 10 μM PNU-120596 by α-CtxArIB[V11L,V16D]. Data points were fitted to the Hill equation, giving IC50 values of 74 ± 1.8 nM and 51.3 ±1.4 nM for choline and AR-R1779, respectively (mean ± SEM, n = 4).

Effects of α-CtxArIB[V11L,V16D] on nicotine- and choline-evoked [3H]dopamine release from rat striatal minces

The ability α-CtxArIB[V11L,V16D] to inhibit nAChR-evoked release of radiolabeled dopamine in vitro was investigated using a high throughput 96 well filter plate assay. BSA (0.01 %) was included in all conditions to prevent non-specific adsorption of α-CtxArIB[V11L,V16D]: BSA had no effect on basal release (Fig 5). Nicotine (10 μM) significantly increased [3H]dopamine release (10.5 ± 2.1 %) compared with buffer alone, and this increase was abolished in the presence of 10 μM mecamylamine. However, α-CtxArIB[V11L,V16D] (300 nM) had no effect on either basal (not shown) or nicotine-evoked [3H]dopamine release (Fig 5A).

Figure 5.

Figure 5

Open in a new tab

Effect of α-CtxArIB[V11L,V16D] on [3H]dopamine release from rat striatal minces evoked by (A) nicotine (Nic) and (B) choline (with or without PNU-120596). Striata were chopped, loaded with [3H]dopamine and release was measured by filtration as described in Materials and Methods. Minces were preincubated for 5 min with or without 300 nM α-CtxArIB[V11L,V16D] (α-Ctx) or 10 μM mecamylamine (Mec). They were then exposed to buffer, 10 μM nicotine (A) or 3 mM choline (B), in the presence or absence of 0.01 % BSA, 10 μM PNU-120596 and/or toxin for a further 5 min before recovery of released [3H]dopamine by filtration. Fractional release of [3H]dopamine was expressed as a percentage of the total in the tissue at the beginning of the stimulation period. Values are mean ± SEM, n = 3 independent experiments, * p < 0.05, significantly different from response to buffer; + p < 0.05, significantly different from evoked release in the absence of α-CtxArIB[V11L,V16D], student’s t-test.

Choline was used to specifically target α7 nAChRs capable of modulating [3H]dopamine release (Fig. 5B). Choline (3 mM) increased fractional release by 6.1 ± 0.9 % and this was further increased in the presence of 10 μM PNU-120596 to 11.9 ± 1.2 %. α-CtxArIB[V11L,V16D] (300 nM) inhibited responses to choline and choline plus PNU-120596 by 81.4 ± 1.2 % and 77.9 ± 1.0 % respectively (Fig 5B).

Discussion

The α-conotoxin ArIB analogue α-CtxArIB[V11L,V16D] potently displaced [125I]α-bungarotoxin binding to rat brain membranes and inhibited functional rat and human α7 nAChR responses in cell lines, transfected oocytes and rat striatal minces. In contrast, α-CtxArIB[V11L,V16D] did not inhibit [3H]-epibatidine binding or functional responses evoked by non-α7 nAChR agonists or KCl. These results confirm the specificity of α-CtxArIB[V11L,V16D] for α7 nAChR over other native nAChRs.

There is increasing evidence for a role for α7 nAChRs in both neural and non-neural signalling and plasticity (McKay et al., 2007; de Jonge and Ulloa, 2007). α7 nAChRs are a therapeutic target for psychiatric and neurological disorders with cognitive or attentional impairments as well as other non-neurological conditions (Mazurov et al., 2006; Chiamulera and Fumagalli, 2007). Investigation of the physiological and pathological roles of α7 nAChRs requires selective ligands. Synthetic efforts in this area have generated several novel α7 nAChR-selective agonists (Jensen et al., 2005; Mazurov et al., 2006; Biton et al., 2007; Hajós et al., 2004) and allosteric modulators (Hurst et al., 2005, Grønlien et al., 2007; Timmermann et al., 2007). However, antagonism of α7 nAChR responses relies on αBgt and MLA, whose utility is constrained by slow kinetics and lack of specificity, respectively. The development of α-CtxArIB[V11L,V16D] and demonstration that it displays high specificity for blockade of α7 nAChRs versus other recombinant nAChRs (including muscle and α9 nAChRs) represents a potentially important advance for α7 nAChR research (Whiteaker et al., 2007). Here we have established that this specificity for α7 nAChRs is preserved with respect to functional native nAChRs and encompasses human as well as rat receptors.

Choline was employed to elicit responses from native α7 nAChRs. Choline is a weak α7 nAChR-selective agonist (Alkondon et al., 1997) and a non-competitive inhibitor of heteromeric nAChRs, including the α3β4 subtype (Alkondon and Albuquerque, 2006). In PC12 and SH-SY5Y cells, the small responses to choline were substantially enhanced by PNU-120596, an allosteric modulator that selectively prolongs agonist-evoked α7 nAChR currents by reducing desensitisation (Hurst et al., 2005; Grønlien et al., 2007). This is in agreement with our previous observations using another α7 nAChR-selective agonist, compound A, in PC12 cells (Dickinson et al., 2007). Similarly, the structurally unrelated α7 nAChR agonist AR-R17779 (Jensen et al., 2005) provoked negligible responses in SH-SY5Y cells when applied alone but in the presence of PNU-120596 significant increases in intracellular Ca2+ resulted. Blockade of the amplified responses by αBgt confirms the specific activation of α7 nAChRs by choline and AR-R17779. α-CtxArIB[V11L,V16D] effectively inhibited choline- and AR-R1779-evoked increases in fluorescence, consistent with its antagonism of α7 nAChRs.

In addition to its potent and selective interaction with rat α7 nAChRs (this study; Whiteaker et al., 2007), α-CtxArIB[V11L,V16D] inhibited recombinant human α7 nAChRs expressed in X. laevis oocytes or native human α7 nAChRs in SH-SY5Y cells. The timecourse of blockade and recovery indicates full inhibition within 2 mins, with a slower recovery taking 15-20 min washout to regain full agonist responsiveness. This rate of recovery is similar to that observed for rat α7 nAChRs in PC12 cells. Although the IC50 values for inhibition of whole cell currents recorded from oocytes expressing recombinant receptors (∼3 nM) were in close agreement for the two species, the IC50 values for inhibition of α7 nAChR-evoked increases in intracellular Ca2+ in both rat and human cell lines were about 20-fold higher (50-80 nM). This is not attributable to differences between agonists used, as choline and AR-R17779 resulted in comparable IC50 values in SH-SY5Y cells. Although the IC50 value for a competitive antagonist is dependent on agonist concentration, all the agonists were applied at a concentration approximating to their EC50 value. Nor is it due to using PNU-120596 to elicit measurable responses from the cell lines, as the IC50 value derived from oocytes was not significantly increased in the presence of the allosteric modulator, in agreement with the lack of effect of PNU-120596 on the ACh inactivation curve (Grønlien et al., 2007). Therefore the consistent discrepancy between recombinant and native preparations is likely to reflect methodological differences between the assays. For example, the apparent potency of competitive antagonists is increased when the agonist duration is very short (Wyllie and Chen, 2007): oocyte recordings used 1 s agonist applications, compared with 20 s for the calcium fluorimetry assays.

Brain α7 nAChRs that modulate striatal [3H]dopamine release from rat striatal minces were also substantially inhibited by 300 nM α-CtxArIB[V11L,V16D]. Although α7 nAChRs are generally accepted to be absent from dopaminergic terminals in the striatum, inhibition of the αBgt-sensitive portion of release by glutamate receptor antagonists supports the localisation of α7 nAChRs on glutamatergic afferents (Kaiser and Wonnacott, 2000). This indirect action is discernable in striatal slices or minces that preserve some local anatomical connections and can be elicited with choline (Barik and Wonnacott, 2006). In the present experiments, PNU-120596 potentiated responses to 1 mM choline and α-CtxArIB[V11L,V16D] was equally effective at blocking responses to choline alone and PNU-120596-potentiated responses.

The specificity of α-CtxArIB[V11L,V16D] for α7 nAChR over other native nAChR subtypes is demonstrated by its failure to inhibit Ca2+ increases elicited by nicotine, 5-I-A85380 or KCl in PC12 cells or nicotine-evoked dopamine release from striatal slices. The inability of α-CtxArIB[V11L,V16D] to displace [3H]epibatidine binding to rat brain membranes confirms that it does not interact with the agonist binding site of non-α7 nAChRs, in agreement with previous binding assays on mouse brain preparations (Whiteaker et al., 2007). In PC12 cells, non-α7 nAChRs comprise α3β2- and α3β4-containing subtypes (Henderson et al., 1994): the lack of any significant blockade by 300 nM α-CtxArIB[V11L,V16D] is consistent with the evaluation of recombinant α3β2 nAChRs in X. laevis oocytes (Whiteaker et al., 2007). Nicotine-evoked [3H]dopamine release is mediated by α4β2- and α6β2-containing nAChRs (Grady et al., 2007). The lack of effect of α-CtxArIB[V11L,V16D] on [3H]dopamine release evoked by 10 μM nicotine contrasts with the modest inhibition of this response by 30 - 100 nM MLA (Mogg et al., 2002). Moreover, MLA selectively inhibits αcontoxin MII-sensitive nAChR-responses on dopaminergic cell bodies (Klink et al., 2001), and this predilection for α6β2-containing nAChRs confounds the use of MLA as an α7 nAChR-selective antagonist in tissue preparations where α6β2* nAChRs might be present. The present evidence for the insensitivity to α-CtxArIB[V11L,V16D] of α4β2- and α6β2-containing nAChRs that mediate striatal dopamine release is supported by its very weak effect on recombinant α6/α3β2β3 nAChRs (IC50 > 20 μM; Whiteaker et al., 2007), giving α-CtxArIB[V11L,V16D] a clear advantage over MLA in this respect. Together with its lack of effect at α9α10 nAChRs and α1 muscle-type nAChRs (see Whiteaker et al., 2007), α-CtxArIB[V11L,V16D] exhibits greater selectivity for α7 nAChRs than either MLA or αBgt.

These findings extend the previous characterisation of α-CtxArIB[V11L,V16D] (Whiteaker et al., 2007) and establishes that it is a specific inhibitor of both rat and human native α7 nAChRs. It exhibits relatively fast blockade and is slowly reversible over several minutes, offering advantages over the very slow kinetics of αBgt inhibition. Its high degree of selectivity, with no effect on α6β2-containing nAChRs or α9 nAChRs, is superior to that of MLA. With effective blockade achieved by sub-micromolar concentrations, α-CtxArIB[V11L,V16D] will be a useful addition to the portfolio of nicotinic ligands.

Acknowledgments

This study was supported by a Medical Research Council UK (MRC) Collaborative PhD Studentship with GlaxoSmithKline (NI); a Biological and Biotechnological Sciences Research Council (BBSRC) PhD studentship (PL); BBSRC grant BBS/B/15600 (SW); NIH grants MH53631 and GM 48677 (JMM) and DA12242 (PW).

Abbreviations

αBgt

αbungarotoxin

α-Ctx

α-conotoxin

α-CtxArIB[V11l,V16D]

α-conotoxin Arenatus IB[V11l,V16D]

AR-R17779

(2)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin]-29-one

BSA

bovine serum albumin

cDNA

complementary DNA

cRNA

complementary RNA

DHβE

dihydro-β-erythroidine

5-I-A85380

5-Iodo-A85380, 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride

KB

Kreb’s buffer

MLA

methyllycaconitine

nAChR

nicotinic acetylcholine receptor

PNU-120596

1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea

TSS

Tyrode’s salt solution

References

  1. Alkondon M, Albuquerque EX. Subtype-specific inhibition of nicotinic acetylcholine receptors by choline: a regulatory pathway. J Pharmacol Exp Ther. 2006;318:268–275. doi: 10.1124/jpet.106.103135. [DOI] [PubMed] [Google Scholar]
  2. Alkondon M, Pereira EF, Cortes WS, Maelicke A, Albuquerque EX. Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons. Eur J Neurosci. 1997;9:2734–2742. doi: 10.1111/j.1460-9568.1997.tb01702.x. [DOI] [PubMed] [Google Scholar]
  3. Baker ER, Zwart R, Sher E, Millar NS. Pharmacological properties of alpha 9 alpha 10 nicotinic acetylcholine receptors revealed by heterologous expression of subunit chimeras. Mol Pharmacol. 2004;65:453–460. doi: 10.1124/mol.65.2.453. [DOI] [PubMed] [Google Scholar]
  4. Barik J, Wonnacott S. Indirect modulation by alpha7 nicotinic acetylcholine receptors of noradrenaline release in rat hippocampal slices: interaction with glutamate and GABA systems and effect of nicotine withdrawal. Mol Pharmacol. 2006;69:618–628. doi: 10.1124/mol.105.018184. [DOI] [PubMed] [Google Scholar]
  5. Biton B, Bergis OE, Galli F, Nedelec A, Lochead AW, Jegham S, Godet D, Lanneau C, Santamaria R, Chesney F, Léonardon J, Granger P, Debono MW, Bohme GA, Sgard F, Besnard F, Graham D, Coste A, Oblin A, Curet O, Vigé X, Voltz C, Rouquier L, Souilhac J, Santucci V, Gueudet C, Françon D, Steinberg R, Griebel G, Oury-Donat F, George P, Avenet P, Scatton B. SSR180711, a novel selective alpha7 nicotinic receptor partial agonist: (1) binding and functional profile. Neuropsychopharmacology. 2007;32:1–16. doi: 10.1038/sj.npp.1301189. [DOI] [PubMed] [Google Scholar]
  6. Blumenthal EM, Conroy WG, Romano SJ, Kassner PD, Berg DK. Detection of functional nicotinic receptors blocked by alpha-bungarotoxin on PC12 cells and dependence of their expression on post-translational events. J Neurosci. 1997;17:6094–7104. doi: 10.1523/JNEUROSCI.17-16-06094.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bschleipfer T, Schukowski K, Weidner W, Grando SA, Schwantes U, Kummer W, Lips KS. Expression and distribution of cholinergic receptors in the human urothelium. Life Sci. 2007;80:2303–2307. doi: 10.1016/j.lfs.2007.01.053. [DOI] [PubMed] [Google Scholar]
  8. Cartier GE, Yoshikami D, Gray WR, Luo S, Olivera BM, McIntosh JM. A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors) J Biol Chem. 1996;271:7522–7528. doi: 10.1074/jbc.271.13.7522. [DOI] [PubMed] [Google Scholar]
  9. Chiamulera C, Fumagalli G. Nicotinic receptors and the treatment of attentional and cognitive deficits in neuropsychiatric disorders: focus on the α7 nicotinic acetylcholine receptor as a promising drug target for schizophrenia. Central Nervous System Agents in Medicinal Chemistry. 2007;7:269–288. [Google Scholar]
  10. Davies AR, Hardick DJ, Blagbrough IS, Potter BV, Wolstenholme AJ, Wonnacott S. Characterisation of the binding of [3H]methyllycaconitine: a new radioligand for labelling alpha 7-type neuronal nicotinic acetylcholine receptors. Neuropharmacology. 1999;38:679–690. doi: 10.1016/s0028-3908(98)00221-4. [DOI] [PubMed] [Google Scholar]
  11. Decker MW, Meyer MD. Therapeutic potential of neuronal nicotinic acetylcholine receptor agonists as novel analgesics. Biochem Pharmacol. 1999;58:917–923. doi: 10.1016/s0006-2952(99)00122-7. [DOI] [PubMed] [Google Scholar]
  12. de Jonge WJ, Ulloa L. The alpha7 nicotinic acetylcholine receptor as a pharmacological target for inflammation. Br J Pharmacol. 2007;151:915–929. doi: 10.1038/sj.bjp.0707264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dickinson JA, Hanrott KE, Mok MH, Kew JN, Wonnacott S. Differential coupling of alpha7 and non-alpha7 nicotinic acetylcholine receptors to calcium-induced calcium release and voltage-operated calcium channels in PC12 cells. J Neurochem. 2007;100:1089–1096. doi: 10.1111/j.1471-4159.2006.04273.x. [DOI] [PubMed] [Google Scholar]
  14. Elgoyhen AB, Vetter DE, Katz E, Rothlin CV, Heinemann SF, Boulter J. alpha10: a determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells. Proc Natl Acad Sci U S A. 2001;98:3501–3506. doi: 10.1073/pnas.051622798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grady SR, Salminen O, Laverty DC, Whiteaker P, McIntosh JM, Collins AC, Marks MJ. The subtypes of nicotinic acetylcholine receptors on dopaminergic terminals of mouse striatum. Biochem Pharmacol. 2007;74:1235–1246. doi: 10.1016/j.bcp.2007.07.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grønlien JH, Håkerud M, Ween H, Thorin-Hagene K, Briggs CA, Gopalakrishnan M, Malysz J. Distinct profiles of alpha7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes. Mol Pharmacol. 2007;72:715–724. doi: 10.1124/mol.107.035410. [DOI] [PubMed] [Google Scholar]
  17. Hajós M, Hurst RS, Hoffmann WE, Krause M, Wall TM, Higdon NR, Groppi VE. The selective alpha7 nicotinic acetylcholine receptor agonist PNU-282987 [N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride] enhances GABAergic synaptic activity in brain slices and restores auditory gating deficits in anesthetized rats. J Pharmacol Exp Ther. 2005;312:1213–1222. doi: 10.1124/jpet.104.076968. [DOI] [PubMed] [Google Scholar]
  18. Henderson LP, Gdovin MJ, Liu C, Gardner PD, Maue RA. Nerve growth factor increases nicotinic ACh receptor gene expression and current density in wild-type and protein kinase A-deficient PC12 cells. J Neurosci. 1994;14:1153–1163. doi: 10.1523/JNEUROSCI.14-03-01153.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hurst RS, Hajós M, Raggenbass M, Wall TM, Higdon NR, Lawson JA, Rutherford-Root KL, Berkenpas MB, Hoffmann WE, Piotrowski DW, Groppi VE, Allaman G, Ogier R, Bertrand S, Bertrand D, Arneric SP. A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization. J Neurosci. 2005;25:4396–4405. doi: 10.1523/JNEUROSCI.5269-04.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jensen AA, Frølund B, Liljefors T, Krogsgaard-Larsen PJ. Neuronal nicotinic acetylcholine receptors: structural revelations, target identifications, and therapeutic inspirations. J Med Chem. 2005;48:4705–4745. doi: 10.1021/jm040219e. [DOI] [PubMed] [Google Scholar]
  21. Kaiser S, Wonnacott S. alpha-bungarotoxin-sensitive nicotinic receptors indirectly modulate [3H]dopamine release in rat striatal slices via glutamate release. Mol Pharmacol. 2000;58:312–318. doi: 10.1124/mol.58.2.312. [DOI] [PubMed] [Google Scholar]
  22. Klink R, de Kerchove d’Exaerde A, Zoli M, Changeux JP. Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei. J Neurosci. 2001;21:1452–1463. doi: 10.1523/JNEUROSCI.21-05-01452.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Levin ED, McClernon FJ, Rezvani AH. Nicotinic effects on cognitive function: behavioral characterization, pharmacological specification, and anatomic localization. Psychopharmacology (Berl) 2006;184:523–539. doi: 10.1007/s00213-005-0164-7. [DOI] [PubMed] [Google Scholar]
  24. Lips KS, König P, Schätzle K, Pfeil U, Krasteva G, Spies M, Haberberger RV, Grando SA, Kummer W. Coexpression and spatial association of nicotinic acetylcholine receptor subunits alpha7 and alpha10 in rat sympathetic neurons. J Mol Neurosci. 2006;30:15–26. doi: 10.1385/JMN:30:1:15. [DOI] [PubMed] [Google Scholar]
  25. Lukas RJ, Norman SA, Lucero L. Characterization of nicotinic acetylcholine receptors expressed by cells of the SH-SY5Y human neuroblastoma clone line. Mol. Cell Neurosci. 1993;4:1–12. doi: 10.1006/mcne.1993.1001. [DOI] [PubMed] [Google Scholar]
  26. Marks MJ, Whiteaker P, Collins AC. Deletion of the alpha7, beta2, or beta4 nicotinic receptor subunit genes identifies highly expressed subtypes with relatively low affinity for [3H]epibatidine. Mol Pharmacol. 2006;70:947–959. doi: 10.1124/mol.106.025338. [DOI] [PubMed] [Google Scholar]
  27. Mazurov A, Hauser T, Miller CH. Selective alpha7 nicotinic acetylcholine receptor ligands. Curr Med Chem. 2006;13:1567–1584. doi: 10.2174/092986706777442011. [DOI] [PubMed] [Google Scholar]
  28. McIntosh JM, Santos AD, Olivera BM. Conus peptides targeted to specific nicotinic acetylcholine receptor subtypes. Ann Rev Biochem. 1999;68:59–88. doi: 10.1146/annurev.biochem.68.1.59. [DOI] [PubMed] [Google Scholar]
  29. McKay BE, Placzek AN, Dani JA. Regulation of synaptic transmission and plasticity by neuronal nicotinic acetylcholine receptors. Biochem Pharmacol. 2007;74:1120–1133. doi: 10.1016/j.bcp.2007.07.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mogg AJ, Whiteaker P, McIntosh JM, Marks M, Collins AC, Wonnacott S. Methyllycaconitine is a potent antagonist of alpha-conotoxin-MII-sensitive presynaptic nicotinic acetylcholine receptors in rat striatum. J Pharmacol Exp Ther. 2002;302:197–204. doi: 10.1124/jpet.302.1.197. [DOI] [PubMed] [Google Scholar]
  31. Nicke A, Wonnacott S, Lewis RJ. Alpha-conotoxins as tools for the elucidation of structure and function of neuronal nicotinic acetylcholine receptor subtypes. Eur J Biochem. 2004;271:2305–2319. doi: 10.1111/j.1432-1033.2004.04145.x. [DOI] [PubMed] [Google Scholar]
  32. Peng X, Katz M, Gerzanich V, Anand R, Lindstrom J. Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes. Mol Pharmacol. 1994;45:546–554. [PubMed] [Google Scholar]
  33. Puttfarcken PS, Jacobs I, Faltynek CR. Characterization of nicotinic acetylcholine receptor-mediated [3H]-dopamine release from rat cortex and striatum. Neuropharmacology. 2000;39:2673–2680. doi: 10.1016/s0028-3908(00)00131-3. [DOI] [PubMed] [Google Scholar]
  34. Ridley DL, Rogers A, Wonnacott S. Differential effects of chronic drug treatment on alpha3* and alpha7 nicotinic receptor binding sites, in hippocampal neurones and SH-SY5Y cells. Br J Pharmacol. 2001;133:1286–1295. doi: 10.1038/sj.bjp.0704207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Timmermann DB, Grønlien JH, Kohlhaas KL, Nielsen EØ, Dam E, Jørgensen TD, Ahring PK, Peters D, Holst D, Christensen JK, Malysz J, Briggs CA, Gopalakrishnan M, Olsen GM. An allosteric modulator of the alpha7 nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivo. J Pharmacol Exp Ther. 2007;323:294–307. doi: 10.1124/jpet.107.120436. [DOI] [PubMed] [Google Scholar]
  36. Wang F, Gerzanich V, Wells GB, Anand R, Peng X, Keyser K, Lindstrom J. Assembly of human neuronal nicotinic receptor alpha5 subunits with alpha3, beta2, and beta4 subunits. J Biol Chem. 1996;271:17656–17665. doi: 10.1074/jbc.271.30.17656. [DOI] [PubMed] [Google Scholar]
  37. Whiteaker P, Christensen S, Yoshikami D, Dowell C, Watkins M, Gulyas J, Rivier J, Olivera BM, McIntosh JM. Discovery, synthesis, and structure activity of a highly selective alpha7 nicotinic acetylcholine receptor antagonist. Biochemistry. 2007;46:6628–6638. doi: 10.1021/bi7004202. [DOI] [PubMed] [Google Scholar]
  38. Whiteaker P, Jimenez M, McIntosh JM, Collins AC, Marks MJ. Identification of a novel nicotinic binding site in mouse brain using [125I]-epibatidine. Br J Pharmacol. 2000;131:729–739. doi: 10.1038/sj.bjp.0703616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wilens TE, Decker MW. Neuronal nicotinic receptor agonists for the treatment of attention-deficit/hyperactivity disorder: focus on cognition. Biochem Pharmacol. 2007;74:1212–1223. doi: 10.1016/j.bcp.2007.07.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wyllie DJ, Chen PE. Taking the time to study competitive antagonism. Br J Pharmacol. 2007;150:541–551. doi: 10.1038/sj.bjp.0706997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Young GT, Broad LM, Zwart R, Astles PC, Bodkin M, Sher E, Millar NS. Species selectivity of a nicotinic acetylcholine receptor agonist is conferred by two adjacent extracellular beta4 amino acids that are implicated in the coupling of binding to channel gating. Mol Pharmacol. 2007;71:389–397. doi: 10.1124/mol.106.030809. [DOI] [PubMed] [Google Scholar]

RESOURCES