Threonine-for-leucine mutation within domain M2 of the neuronal alpha(7) nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist.

A study was made of the effects of 5-hydroxytryptamine (5HT) on homomeric neuronal nicotinic receptors (nAcChoR) expressed in Xenopus oocytes after injection of cDNA encoding the wild-type chicken alpha(7) subunit. Acetylcholine (AcCho) elicited large currents (IAcCho) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC50) of 56 microM and a Hill coefficient (nH) of 1.2. The inhibition of IAcCho by 5HT was noncompetitive and voltage independent, a behavior incompatible with a channel blockade mechanism. 5HT alone did not elicit membrane currents in oocytes injected with the wild-type alpha(7) subunit cDNA. In contrast, 5HT elicited membrane currents (I5HT) in oocytes injected with cDNA encoding an alpha(7) mutant subunit with a threonine-for-leucine-247 substitution (L247T alpha(7)). I5HT was inhibited by the potent nicotinic receptor blockers alpha-bungarotoxin (100 nM) and methyllycaconitine (1 microM). Furthermore, the characteristics of I5HT, including its voltage dependence, were similar to those of IAcCho. The 5HT dose-I5HT response gave an apparent dissociation constant EC50 of 23.5 microM and a Hill coefficient nH of 1.7, which were not modified by the presence of AcCho. Similarly, the apparent affinity of L247T alpha(7) for AcCho as well as its cooperativity were not influenced by 5HT, indicating a lack of mutual interactions between 5HT and AcCho. These results show that 5HT is a potent noncompetitive antagonist of neuronal alpha(7) nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2.

Role of alpha 7 Nicotinic Acetylcholine Receptor in Calcium Signaling Induced by Prion Protein Interaction with Stress-inducible Protein 1

The prion protein (PrP(C)) is a conserved glycosylphosphatidyl-inositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP(C) extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrPC-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP(C) engagement induces an increase in intracellular Ca(2+) levels. This effect was not detected in PrP(C)-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP(C). Using a best candidate approach to test for potential channels involved in Ca(2+) influx evoked by STI1-PrP(C), we found that alpha-bungarotoxin, a specific inhibitor for alpha 7 nicotinic acetylcholine receptor (alpha 7nAChR), was able to block PrP(C)-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when alpha 7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP(C) and allowed reconstitution of signaling by PrP(C)-STI1 interaction. These results indicate that STI1 can interact with the PrP(C).alpha 7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.

Strychnine activates neuronal α7 nicotinic receptors after mutations in the leucine ring and transmitter binding site domains

Recent work has shown that strychnine, the potent and selective antagonist of glycine receptors, is also an antagonist of nicotinic acetylcholine (AcCho) receptors including neuronal homomeric α7 receptors, and that mutating Leu-247 of the α7 nicotinic AcCho receptor-channel domain (L247Tα7; mut1) converts some nicotinic antagonists into agonists. Therefore, a study was made of the effects of strychnine on Xenopus oocytes expressing the chick wild-type α7 or L247Tα7 receptors. In these oocytes, strychnine itself did not elicit appreciable membrane currents but reduced the currents elicited by AcCho in a reversible and dose-dependent manner. In sharp contrast, in oocytes expressing L247Tα7 receptors with additional mutations at Cys-189 and Cys-190, in the extracellular N-terminal domain (L247T/C189–190Sα7; mut2), micromolar concentrations of strychnine elicited inward currents that were reversibly inhibited by the nicotinic receptor blocker α-bungarotoxin. Single-channel recordings showed that strychnine gated mut2-channels with two conductance levels, 56 pS and 42 pS, and with kinetic properties similar to AcCho-activated channels. We conclude that strychnine is a modulator, as well as an activator, of some homomeric nicotinic α7 receptors. After injecting oocytes with mixtures of cDNAs encoding mut1 and mut2 subunits, the expressed hybrid receptors were activated by strychnine, similar to the mut2, and had a high affinity to AcCho like the mut1. A pentameric symmetrical model yields the striking conclusion that two identical α7 subunits may be sufficient to determine the functional properties of α7 receptors.

Alpha-selenoconotoxins, a new class of potent alpha(7) neuronal nicotinic receptor antagonists

Disulfide bonds are important structural motifs that play an essential role in maintaining the conformational stability of many bioactive peptides. Of particular importance are the conotoxins, which selectively target a wide range of ion channels that are implicated in numerous disease states. Despite the enormous potential of conotoxins as therapeutics, their multiple disulfide bond frameworks are inherently unstable under reducing conditions. Reduction or scrambling by thiol-containing molecules such as glutathione or serum albumin in intracellular or extracellular environments such as blood plasma can decrease their effectiveness as drugs. To address this issue, we describe a new class of selenoconotoxins where cysteine residues are replaced by selenocysteine to form isosteric and non-reducible diselenide bonds. Three isoforms of alpha-conotoxin ImI were synthesized by t-butoxycarbonyl chemistry with systematic replacement of one([ Sec(2,8)] ImI or [Sec(3,12)] ImI), or both([Sec(2,3,8,12)] ImI) disulfide bonds with a diselenide bond. Each analogue demonstrated remarkable stability to reduction or scrambling under a range of chemical and biological reducing conditions. Three-dimensional structural characterization by NMR and CD spectroscopy indicates conformational preferences that are very similar to those of native ImI, suggesting fully isomorphic structures. Additionally, full bioactivity was retained at the alpha(7) nicotinic acetylcholine receptor, with each seleno-analogue exhibiting a dose-response curve that overlaps with wild-type ImI, thus further supporting an isomorphic structure. These results demonstrate that selenoconotoxins can be used as highly stable scaffolds for the design of new drugs.

alpha-Conotoxin ImI inhibits the alpha-bungarotoxin-resistant nicotinic response in bovine adrenal chromaffin cells

The activity of alpha-conotoxin (alpha-CTX) lml, from the vermivorous marine snail Conus imperialis, has been studied on mammalian nicotinic receptors on bovine chromaffin cells and at the rat neuromuscular junction. Synthetic alpha-CTX lml was a potent inhibitor of the neuronal[ nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 mu M, log IC50 = 0.4 +/- 0.07), showing competitive inhibition of nicotine-evoked catecholamine secretion. (alpha-CTX lml also inhibited nicotine-evoked Ca-45(2+) uptake but not Ca-45(2+) uptake stimulated by 56 mM Kr. In contrast, alpha-CTX lml had no effect at the neuromuscular junction over the concentration range 1-20 mu M. Bovine chromaffin cells are known to contain the alpha 3 beta 4, alpha 7, and (possibly) alpha 3 beta 4 alpha 5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by alpha-bungarotoxin, indicating that alpha 7 nicotinic receptors are not involved. We propose that alpha-CTX lml interacts selectively with the functional (alpha 3 beta 4 or alpha 3 beta 4 alpha 5) nicotinic acetylcholine receptor to inhibit the neuronal-type nicotinic response in bovine chromaffin cells.

Chronic infusion of amyloid-β peptide and sustained attention altered α7 nicotinic receptor density in the rat brain

It is already known that progressive degeneration of cholinergic neurons in brain areas such as the hippocampus and the cortex leads to memory deficits, as observed in Alzheimer's disease. This work verified the effects of the infusion of amyloid-beta (A beta) peptide associated to an attentional rehearsal on the density of alpha 7 nicotinic cholinergic receptor (nAChR) in the brain of male Wistar rats. Animals received intracerebroventricular infusion of A beta or vehicle (control - C) and their attention was stimulated weekly (Stimulated A beta group: S-A beta and Stimulated Control group: SC) or not (Non-Stimulated A beta group: N-SA beta and Non-Stimulated Control group: N-SC), using an active avoidance apparatus. Conditioned avoidance responses (CAR) were registered. Chronic infusion of A beta caused a 37% reduction in CAR for N-SA beta. In S-A beta, this reduction was not observed. At the end, brains were extracted and autoradiography for alpha 7 nAChR was conducted using [I-125]-alpha-bungarotoxin. There was an increase in alpha 7 density in hippocampus, cortex and amygdala of SA beta animals, together with the memory preservation. In recent findings from our lab using mice infused with A beta and the alpha 7 antagonist methyllycaconitine, and stimulated weekly in the same apparatus, it was observed that memory maintenance was abolished. So, the increase in alpha 7 density in brain areas related to memory might be related to a participation of this receptor in the long-lasting change in synaptic plasticity, which is important to improve and maintain memory consolidation.

Heteromeric nicotinic receptors are involved in the sensitization and addictive properties of MDMA in mice

We have investigated the effect of nicotinic receptor ligands in the behavioral sensitization (hyperlocomotion) and rewarding properties (conditioned place preference paradigm, CPP) of 3,4-methylenedioxy-methamphetamine (MDMA) in mice. Each animal received intraperitoneal pretreatment with either saline, dihydro-β-erythroidine (DHβE, 1 mg/kg) or varenicline (VAR, 0.3 mg/kg), 15 min prior to subcutaneous saline or MDMA (5 mg/kg), for 10 consecutive days. On day 1, both DHβE and VAR inhibited the MDMA-induced hyperlocomotion. After 10 days of treatment, MDMA induced a hyperlocomotion that was not reduced (rather enhanced) in antagonist-pretreated animals. This early hyperlocomotion was accompanied by a significant increase in heteromeric nicotinic receptors in cortex that was not blocked by DHβE or VAR. Behavioral sensitization to MDMA was highest 2 weeks after the discontinuation of MDMA treatment. This additional increase in sensitivity was prevented in animals pretreated with DHβE or VAR. At this time, MDMA-treated mice showed a significant increase in heteromeric receptors in cortex that was prevented by DHβE and VAR. An involvement of α7 nicotinic receptors in this effect is ruled out. MDMA (10 mg/kg) induced positive CPP that was abolished by DHβE (2 mg/kg) and VAR (2 mg/kg). Moreover, chronic nicotine pretreatment (2 mg/kg, ip, b.i.d., for 14 days) caused MDMA, administered at a low dose (3 mg/kg), to induce CPP, which would otherwise not occur. Finally, present results point out that heteromeric nicotinic receptors are involved in locomotor sensitization and addictive potential induced by MDMA. Thus, varenicline might be a useful drug to treat both tobacco and MDMA abuse at once.

Heteromeric nicotinic receptors are involved in the sensitization and addictive properties of MDMA in mice

We have investigated the effect of nicotinic receptor ligands in the behavioral sensitization (hyperlocomotion) and rewarding properties (conditioned place preference paradigm, CPP) of 3,4-methylenedioxy-methamphetamine (MDMA) in mice. Each animal received intraperitoneal pretreatment with either saline, dihydro-β-erythroidine (DHβE, 1 mg/kg) or varenicline (VAR, 0.3 mg/kg), 15 min prior to subcutaneous saline or MDMA (5 mg/kg), for 10 consecutive days. On day 1, both DHβE and VAR inhibited the MDMA-induced hyperlocomotion. After 10 days of treatment, MDMA induced a hyperlocomotion that was not reduced (rather enhanced) in antagonist-pretreated animals. This early hyperlocomotion was accompanied by a significant increase in heteromeric nicotinic receptors in cortex that was not blocked by DHβE or VAR. Behavioral sensitization to MDMA was highest 2 weeks after the discontinuation of MDMA treatment. This additional increase in sensitivity was prevented in animals pretreated with DHβE or VAR. At this time, MDMA-treated mice showed a significant increase in heteromeric receptors in cortex that was prevented by DHβE and VAR. An involvement of α7 nicotinic receptors in this effect is ruled out. MDMA (10 mg/kg) induced positive CPP that was abolished by DHβE (2 mg/kg) and VAR (2 mg/kg). Moreover, chronic nicotine pretreatment (2 mg/kg, ip, b.i.d., for 14 days) caused MDMA, administered at a low dose (3 mg/kg), to induce CPP, which would otherwise not occur. Finally, present results point out that heteromeric nicotinic receptors are involved in locomotor sensitization and addictive potential induced by MDMA. Thus, varenicline might be a useful drug to treat both tobacco and MDMA abuse at once.

alpha-conotoxins EpI and AuIB switch subtype selectivity and activity in native versus recombinant nicotinic acetylcholine receptors

The Xenopus laevis oocyte expression system was used to determine the activities of alpha-conotoxins EpI and the ribbon isomer of AuIB, on defined nicotinic acetylcholine receptors (nAChRs). In contrast to previous findings on intracardiac ganglion neurones, alpha-EpI showed no significant activity on oocyte-expressed alpha3beta4 and alpha3beta2 nAChRs but blocked the alpha7 nAChR with an IC50 value of 30 nM. A similar IC50 value (103 nM) was obtained on the alpha7/5HT(3) chimeric receptor stably expressed in mammalian cells. Ribbon AuIB maintained its selectivity on oocyte-expressed alpha3beta4 receptors but unlike in native cells, where it was 10-fold more potent than native alpha-AuIB, had 25-fold lower activity. These results indicate that as yet unidentified factors influence alpha-conotoxin pharmacology at native versus oocyte-expressed nAChRs. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

alpha-conotoxins PnIA and [A10L] PnIA stabilize different states of the alpha 7-L247T nicotinic acetylcholine receptor

The effects of the native alpha-conotoxin PnIA, its synthetic derivative [ A10L] PnIA and alanine scan derivatives of [ A10L] PnIA were investigated on chick wild type alpha7 and alpha7-L247T mutant nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. PnIA and [A10L] PnIA inhibited acetylcholine (ACh)-activated currents at wtalpha7 receptors with IC50 values of 349 and 168 nM, respectively. Rates of onset of inhibition were similar for PnIA and [ A10L] PnIA; however, the rate of recovery was slower for [ A10L] PnIA, indicating that the increased potency of [ A10L] PnIA at alpha7 receptors is conveyed by its slower rate of dissociation from the receptors. All the alanine mutants of [ A10L] PnIA inhibited ACh-activated currents at wtalpha7 receptors. Insertion of an alanine residue between position 5 and 13 and at position 15 significantly reduced the ability of [ A10L] PnIA to inhibit ACh-evoked currents. PnIA inhibited the non-desensitizing ACh-activated currents at alpha7-L247T receptors with an IC50 194 nM. In contrast, [ A10L] PnIA and the alanine mutants potentiated the ACh-activated current alpha7-L247T receptors and in addition [ A10L] PnIA acted as an agonist. PnIA stabilized the receptor in a state that is non-conducting in both the wild type and mutant receptors, whereas [ A10L] PnIA stabilized a state that is non-conducting in the wild type receptor and conducting in the alpha7-L247T mutant. These data indicate that the change of a single amino acid side-chain, at position 10, is sufficient to change the toxin specificity for receptor states in the alpha7-L247T mutant.

Molecule modeling of human pentameric alpha(7) neuronal nicotinic acetylcholine receptor and its interaction with its agonist and competitive antagonist

The nicotinic Acetylcholine Receptor (nAChR) is the major class of neurotransmitter receptors that is involved in many neurodegenerative conditions such as schizophrenia, Alzheimer's and Parkinson's diseases. The N-terminal region or Ligand Binding Domain (LBD) of nAChR is located at pre- and post-synaptic nervous system, which mediates synaptic transmission. nAChR acts as the drug target for agonist and competitive antagonist molecules that modulate signal transmission at the nerve terminals. Based on Acetylcholine Binding Protein (AChBP) from Lymnea stagnalis as the structural template, the homology modeling approach was carried out to build three dimensional model of the N-terminal region of human alpha(7)nAChR. This theoretical model is an assembly of five alpha(7) subunits with 5 fold axis symmetry, constituting a channel, with the binding picket present at the interface region of the subunits. alpha-netlrotoxin is a potent nAChR competitive antagonist that readily blocks the channel resulting in paralysis. The molecular interaction of alpha-Bungarotoxin, a long chain alpha-neurotoxin from (Bungarus multicinctus) and human alpha(7)nAChR seas studied. Agonists such as acetylcholine, nicotine, which are used in it diverse array of biological activities, such as enhancements of cognitive performances, were also docked with the theoretical model of human alpha(7)nAChR. These docked complexes were analyzed further for identifying the crucial residues involved in interaction. These results provide the details of interaction of agonists and competitive antagonists with three dimensional model of the N-terminal region of human alpha(7)nAChR and thereby point to the design of novel lead compounds.

Neuronal nicotinic threonine-for-leucine 247 α7 mutant receptors show different gating kinetics when activated by acetylcholine or by the noncompetitive agonist 5-hydroxytryptamine

Mutation of the highly conserved leucine residue (Leu-247) converts 5-hydroxytryptamine (5HT) from an antagonist into an agonist of neuronal homomeric α7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We show here that acetylcholine (AcCho) activates two classes of single channels with conductances of 44 pS and 58 pS, similar to those activated by 5HT. However, the mean open time of AcCho-gated ion channels (11 ms) is briefer than that of 5HT-gated ion channels (18 ms). Furthermore, whereas the open time of AcCho channels lengthens with hyperpolarization, that of 5HT channels is decreased. In voltage-clamped oocytes, the apparent affinity of the α7 mutant receptor for 5HT is not modified by the presence of dihydro-β-erythroidine, which acts on the AcCho binding site in a competitive manner. This indicates a noncompetitive action of 5HT on nicotinic acetylcholine receptors. Considered together, our findings show that AcCho gates α7 mutant channels with similar conductance but with different kinetic profile than the channels gated by 5HT, suggesting that the two agonists act on different docking sites. These results will help to understand the crosstalk between cholinergic and serotonergic systems in the central nervous system.

β-Amyloid peptide blocks the response of α7-containing nicotinic receptors on hippocampal neurons

Alzheimer's disease produces a devastating decline in mental function, with profound effects on learning and memory. Early consequences of the disease include the specific loss of cholinergic neurons in brain, diminished cholinergic signaling, and the accumulation of β-amyloid peptide in neuritic plaques. Of the nicotinic acetylcholine receptors at risk, the most critical may be those containing the α7 gene product (α7-nAChRs), because they are widespread, have a high relative permeability to calcium, and regulate numerous cellular events in the nervous system. With the use of whole-cell patch–clamp recording we show here that nanomolar concentrations of β-amyloid peptides specifically and reversibly block α7-nAChRs on rat hippocampal neurons in culture. The block is noncompetitive, voltage-independent, and use-independent and is mediated through the N-terminal extracellular domain of the receptor. It does not appear to require either calcium influx or G protein activation. β-Amyloid blockade is likely to be a common feature of α7-nAChRs because it applies to the receptors at both somato-dendritic and presynaptic locations on rat hippocampal neurons and extends to homologous receptors on chick ciliary ganglion neurons as well. Because α7-nAChRs in the central nervous system are thought to have numerous functions and recently have been implicated in learning and memory, impaired receptor function in this case may contribute to cognitive deficits associated with Alzheimer's disease.