Alpha7 nAChR Research Articles

Dopamine D 2 ‐receptor activation elicits akinesia, rigidity, catalepsy, and tremor in mice expressing hypersensitive α4 nicotinic receptors via a cholinergic‐dependent mechanism

Recent studies suggest that high-affinity neuronal nicotinic acetylcholine receptors (nAChRs) containing alpha4 and beta2 subunits (alpha4beta2*) functionally interact with G-protein-coupled dopamine (DA) D(2) receptors in basal ganglia. We hypothesized that if a functional interaction between these receptors exists, then mice expressing an M2 point mutation (Leu9'Ala) rendering alpha4 nAChRs hypersensitive to ACh may exhibit altered sensitivity to a D(2)-receptor agonist. When challenged with the D(2)R agonist, quinpirole (0.5-10 mg/kg), Leu9'Ala mice, but not wild-type (WT) littermates, developed severe, reversible motor impairment characterized by rigidity, catalepsy, akinesia, and tremor. While striatal DA tissue content, baseline release, and quinpirole-induced DA depletion did not differ between Leu9'Ala and WT mice, quinpirole dramatically increased activity of cholinergic striatal interneurons only in mutant animals, as measured by increased c-Fos expression in choline acetyltransferase (ChAT)-positive interneurons. Highlighting the importance of the cholinergic system in this mouse model, inhibiting the effects of ACh by blocking muscarinic receptors, or by selectively activating hypersensitive nAChRs with nicotine, rescued motor symptoms. This novel mouse model mimics the imbalance between striatal DA/ACh function associated with severe motor impairment in disorders such as Parkinson's disease, and the data suggest that a D(2)R-alpha4*-nAChR functional interaction regulates cholinergic interneuron activity.

Mechanisms of Neuroprotective Effects of Nicotine and Acetylcholinesterase Inhibitors: Role of α4 and α7 Receptors in Neuroprotection

Neurotoxicity induced by glutamate and other excitatory amino acids has been implicated in various neurodegenerative disorders including hypoxic ischemic events, trauma, and Alzheimer's and Parkinson's diseases. We examined the roles of nicotinic acetylcholine receptors (nAChRs) in survival of CNS neurons during excitotoxic events. Nicotine as well as other nicotinic receptor agonists protected cortical neurons against glutamate neurotoxicity via alpha4 and alpha7 nAChRs at least partly by inhibiting the process of apoptosis in near-pure neuronal cultures obtained from the cerebral cortex of fetal rats. Donepezil, galanatamine and tacrine, therapeutic acetylcholinesterase (AChE) inhibitors currently being used for treatment of Alzheimer's disease also protected neuronal cells from glutamate neurotoxicity. Protective effects of nicotine and the AChE inhibitors were antagonized by nAChR antagonists. Moreover, nicotine and those AChE inhibitors induced up-regulation of nAChRs. Inhibitors for a non-receptor-type tyrosine kinase, Fyn, and janus-activated kinase 2, suppressed the neuroprotective effect of donepezil and galantamine. Furthermore, a phosphatidylinositol 3-kinase (PI3K) inhibitor also suppressed the neuroprotective effect of the AChE inhibitors. The phosphorylation of Akt, an effector of PI3K, and the expression level of Bcl-2, an anti-apoptotic protein, increased with donepezil and galantamine treatments. These results suggest that nicotine as well as AChE inhibitors, donepezil and galantamine, prevent glutamate neurotoxicity through alpha4 and alpha7 nAChRs and the PI3K-Akt pathway.

The Ubiquitin–Proteasome System Regulates the Stability of Neuronal Nicotinic Acetylcholine Receptors

Ubiquitination is a key event for protein degradation by the proteasome system, membrane protein internalization, and protein trafficking among cellular compartments. Few data are available on the role of the ubiquitin-proteasome system (UPS) in the trafficking of neuronal nicotinic acetylcholine receptors (nAChRs). Experiments conducted in neuron-like differentiated rat pheochromocytoma cells (PC12 cells) show that the alpha3, beta2, and beta4 nAChR subunits are ubiquitinated and that their ubiquitination is necessary for degradation. A 24-h treatment with the proteasome inhibitor PS-341 increased the total levels of alpha3 and the two beta subunits in both whole cell lysates and fractions enriched for the ER/Golgi compartment. nAChR subunit upregulation was also detected in plasma membrane-enriched fractions. Inhibition of the lysosomal degradation machinery by E-64 had a significantly smaller effect on nAChR turnover. The present data, together with previous results showing that the alpha7 nAChR subunit is a target of the UPS, point to a prominent role of the proteasome in nAChR trafficking.

Glutamate–Dopamine Crosstalk in the Rat Prefrontal Cortex is Modulated by Alpha7 Nicotinic Receptors and Potentiated by PNU-120596

The aim of this study was to explore the modulation by alpha7 nicotinic receptors (nAChRs) of dopamine and glutamate release in the rat prefrontal cortex where these receptors are implicated in attentional processes and are therapeutic targets for cognitive deficits. The presence of presynaptic alpha7 nAChRs on glutamate terminals is supported by the ability of the subtype-selective agonist Compound A to evoke [(3)H]D-aspartate release from synaptosomes: This response was potentiated by the selective allosteric potentiator PNU-120596 and blocked by alphabungarotoxin. Compound A also evoked dopamine overflow in the prefrontal cortex in vivo, and this was potentiated by PNU-120596. alpha7 nAChR-evoked [(3)H]dopamine release from tissue prisms in vitro was blocked by antagonists of NMDA and AMPA receptors. These data are consistent with a model in which alpha7 nAChRs present on glutamate terminals increase glutamate release that (1) contributes to presynaptic facilitation and synaptic plasticity and (2) co-ordinately enhances dopamine release from neighbouring boutons.

Haeme oxygenase-1 overexpression via nAChRs and the transcription factor Nrf2 has antinociceptive effects in the formalin test

Epibatidine has shown antinociceptive effects in various pain models, being 200-fold more potent than morphine. Previous results from our laboratory demonstrated that HO-1 overexpression has an antinociceptive effect in the formalin test. Furthermore, epibatidine was able to induce haeme oxygenase-1 (HO-1). So, the aim of this study was to investigate the effect of HO-1 overexpression induced by epibatidine in nociception elicited by formalin injection in the mice hindpaw. Administration of epibatidine (4 μg/kg) 24 h before the test reduced the nociceptive response during the first phase and second phase of the formalin test. This effect was prevented by treatment with tin protoporphyrin (SnPP, an inhibitor of HO-1 activity) administered via intraplantar 5 min before the test, suggesting a main role of HO-1. Western blot analysis revealed that epibatidine treatment increased by 2-fold HO-1 expression in the paw; this effect was lost in knockout mice for nuclear factor-erythroid 2-related factor 2 (Nrf2) and was accompanied by the loss of its antinociceptive effect. Furthermore, the antinociceptive effect of epibatidine was related to the activation of alpha7 and/or alpha9 nAChRs since methyllycaconitine (MLA) and mecamylamine but not dihydro-β-erythroidine (DHβE) reverted this effect. Finally, we showed by flow cytometry and by immunofluorescence that white blood cells of the animals injected with epibatidine expressed more HO-1 than control animals, and this expression was also reverted by MLA pre-treatment. These findings demonstrate that HO-1 induction by epibatidine has antinociceptive and anti-inflammatory effects by the activation of MLA-sensitive nAChRs.

Prenatal nicotine exposure alters medullary nicotinic and AMPA-mediated control of respiratory frequency in vitro

Prenatal nicotine exposure (PNE) is correlated with breathing abnormalities in humans and other animals. Despite evidence that this relationship results from alterations in nicotinic acetylcholine receptors (nAChRs), the mechanisms are poorly understood. Here, we hypothesize that PNE blunts nAChR-mediated respiratory-related motor output. We also hypothesize that the PNE-induced changes in nAChRs leads to secondary alterations in glutamatergic neurotransmission. To test these hypotheses, we used an in vitro brainstem-spinal cord preparation and recorded C4 ventral root (C4 VR) nerve bursts from 0 to 4-day-old rats that were exposed to either nicotine (6mgkg(-1)day(-1)) or saline (control) in utero. Nicotine bitartrate, nAChR antagonists, NMDA and AMPA were applied to the brainstem compartment of a "split-bath" configuration, which physically separated the medulla from the spinal cord. Nicotine (0.2 or 0.5microM) increased peak C4 VR burst frequency by over 230% in control pups, but only 140% in PNE animals. The application of nAChR antagonists showed that these effects were mediated by the alpha4beta2 nAChR subtype with no effect on alpha7 nAChRs in either group. We also show that AMPA-mediated excitatory neurotransmission is enhanced by PNE, but NMDA-mediated neurotransmission is unaltered. These data and the work of others suggest that the PNE may functionally desensitize alpha4beta2 nAChRs located on the presynaptic terminals of glutamatergic neurons leading to less neurotransmitter release, which in turn up-regulates AMPA receptors on rhythm generating neurons.

Evaluation of α7 Nicotinic Acetylcholine Receptor Agonists and Positive Allosteric Modulators Using the Parallel Oocyte Electrophysiology Test Station

Neuronal acetylcholine receptors (nAChRs) of the alpha7 subtype are ligand-gated ion channels that are widely distributed throughout the central nervous system and considered as attractive targets for the treatment of various neuropsychiatric and neurodegenerative diseases. Both agonists and positive allosteric modulators (PAMs) are being developed as means to enhance the function of alpha7 nAChRs. The in vitro characterization of alpha7 ligands, including agonists and PAMs, relies on multiple technologies, but only electrophysiological measurements assess the channel activity directly. Traditional electrophysiological approaches utilizing two-electrode voltage clamp or patch clamp in isolated cells have very low throughput to significantly impact drug discovery. Abbott (Abbott Park, IL) has developed a two-electrode voltage clamp-based system, the Parallel Oocyte Electrophysiology Test Station (POETs()), that allows for the investigation of ligand-gated ion channels such as alpha7 nAChRs in a higher-throughput manner. We describe the utility of this technology in the discovery of selective alpha7 agonists and PAMs. With alpha7 agonists, POETs experiments involved both single- and multiple-point concentration-response testing revealing diverse activation profiles (zero efficacy desensitizing, partial, and full agonists). In the characterization of alpha7 PAMs, POETs testing has served as a reliable primary or secondary screen identifying compounds that fall into distinct functional types depending on the manner in which current potentiation occurred. Type I PAMs (eg, genistein, NS1738, and 5-hydroxyindole) increase predominantly the peak amplitude response, type II PAMs affect the peak current and current decay (eg, PNU-120,596 and 4-(naphthalen-1-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), and anothertype slowing the current decay kinetics in the absence of increases in the peak current. In summary, POETs technology allows for significant impact on higher throughput in the testing of alpha7 agonists and PAMs and for identification of compounds with unique profiles that could prove valuable in identifying an optimum in vitro profile in the development of therapeutics for which the alpha7 subtype is considered.

Coupling of Ionic Events to Protein Kinase Signaling Cascades upon Activation of α7 Nicotinic Receptor: COOPERATIVE REGULATION OF α2-INTEGRIN EXPRESSION AND Rho KINASE ACTIVITY

Defining the signaling mechanisms and effector proteins mediating phenotypic and mechanical plasticity of keratinocytes (KCs) during wound epithelialization is one of the major goals in epithelial cell biology. The acetylcholine (ACh)-gated ion channels, or nicotinic ACh receptors (nAChRs), mediate the nicotinergic signaling that controls crawling locomotion of KCs. To elucidate relative contributions of the ionic and protein kinase-mediated events elicited due to activation of alpha7 nAChRs, we quantitated expression of alpha2-integrin gene at the mRNA and protein levels and also measured Rho kinase activity in KCs stimulated with the alpha7 agonist AR-R17779 while blocking the Na+ or Ca2+ entry and/or inhibiting signaling kinases. The results demonstrated the existence of the two-component signaling systems coupling the ionic events and protein kinase signaling cascades downstream of alpha7 nAChR to simultaneous up-regulation of alpha2-integrin expression and activation of Rho kinase. The Raf/MEK1/ERK1/2 cascade up-regulating alpha2-integrin was activated due to both Ca2+-dependent recruitment of Ca2+/calmodulin-dependent protein kinase II and protein kinase C and Ca2+-independent activation of Ras. Likewise the phosphatidylinositol 3-kinase-mediated activation of Rho kinase was elicited due to both Ca2+ entry-dependent involvement of Ca2+/calmodulin-dependent protein kinase II and Ca2+-independent activation of Jak2. Thus, although the initial signals emanating from activated alpha7 nAChR are different in nature the pathways intersect at common effector molecules providing for a common end point effect. This novel paradigm of nAChR-mediated coordination of the ionic and metabolic signaling events can allow an auto/paracrine ACh to simultaneously alter gene expression and induce reciprocal changes in the cytoskeleton and contractile system of KCs required to compete a particular step of wound epithelialization.

The Role of α6-Containing Nicotinic Acetylcholine Receptors in Nicotine Reward and Withdrawal

The alpha6 nicotinic acetylcholine receptor (nAChR) subunit is involved in nicotine-stimulated dopamine release in the striatum. It is expressed in brain regions and coexpressed with nAChR subtypes implicated in nicotine dependence behaviors; hence, this subunit may play a role in nicotine dependence. Using the alpha6-selective antagonist alpha-conotoxin H9A;L15A (MII[H9A;L15A]), we determined the role of alpha6* nAChRs in the pharmacological and behavioral effects of nicotine. We measured effects of pretreatment with MII[H9A;L15A] on analgesia, locomotion, and body temperature after a single injection of nicotine. Effects of MII[H9A;L15A] on nicotine reward were measured using the conditioned place preference (CPP) paradigm. We further measured physical (somatic signs and hyperalgesia) and affective [anxiety-related behavior and conditioned place aversion (CPA)] nicotine withdrawal behaviors after extended nicotine exposure. Results showed that MII[H9A;L15A] did not block acute nicotine effects on the behaviors measured. Conversely, MII[H9A:l15A] blocked the expression of nicotine CPP, as well as withdrawal-associated CPA and anxiety-related behavior in the elevated plus maze, but not withdrawal-induced somatic signs or hyperalgesia. These results suggest a role for the alpha6 nAChR subunit in nicotine reward and affective nicotine withdrawal but not acute nicotine-induced or physical withdrawal behaviors.

Differential contribution of genetic variation in multiple brain nicotinic cholinergic receptors to nicotine dependence: recent progress and emerging open questions

Nicotine dependence (ND), a major public health challenge, is a complex, multifactorial behavior, in which both genetic and environmental factors have a role. Brain nicotinic acetylcholine receptor (nAChR)-encoding genes are among the most prominent candidate genes studied in the context of ND, because of their biological relevance as binding sites for nicotine. Until recently, most research on the role of nAChRs in ND has focused on two of these genes (encoding the alpha4- and beta2-subunits) and not much attention has been paid to the possible contribution of the other nine brain nAChR subunit genes (alpha2-alpha3, alpha5-alpha7, alpha9-alpha10, beta3-beta4) to the pathophysiology and genetics of ND. This situation has changed dramatically in the last 2 years during which intensive research had addressed the issue, mainly from the genetics perspective, and has shown the importance of the CHRNA5-CHRNA3-CHRNB4 and CHRNA6-CHRNB3 loci in ND-related phenotypes. In this review, we highlight recent findings regarding the contribution of non-alpha4/beta2-subunit containing nAChRs to ND, based on several lines of evidence: (1) human genetics studies (including linkage analysis, candidate-gene association studies and whole-genome association studies) of several ND-related phenotypes; (2) differential pharmacological and biochemical properties of receptors containing these subunits; (3) evidence from genetically manipulated mice; and (4) the contribution of nAChR genes to ND-related personality traits and neurocognitive profiles. Combining neurobiological genetic and behavioral perspectives, we suggest that genetic susceptibility to ND is not linked to one or two specific nAChR subtype genes but to several. In particular, the alpha3, alpha5-6 and beta3-4 nAChR subunit-encoding genes may play a much more pivotal role in the neurobiology and genetics of ND than was appreciated earlier. At the functional level, variants in these subunit genes (most likely regulatory) may have independent as well as interactive contributions to the ND phenotype spectrum. We address methodological challenges in the field, highlight open questions and suggest possible pathways for future research.

Optimizing the therapeutic targeting of alpha7 nAChR for the treatment of Alzheimer's disease

Optimizing the therapeutic targeting of alpha7 nAChR for the treatment of Alzheimer's disease

Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR alpha7 subunit without compromising formation of functional receptors. A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplasmic loop between the third and fourth transmembrane domains of the mouse nAChR alpha7 subunit (alpha7Y). SH-EP1 cells were transfected with mouse nAChR wild type alpha7 subunits (alpha7) or with alpha7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with (125)I-labeled alpha-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy. Whole-cell currents revealed that alpha7Y nAChRs and alpha7 nAChRs were functional with comparable EC(50) values for the alpha7 nAChR-selective agonist, choline, and IC(50) values for the alpha7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that alpha7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little alpha7Y localized to the plasma membrane, typical of alpha7 nAChRs. nAChRs composed as homooligomers of alpha7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of alpha7 nAChRs. alpha7Y nAChRs may be used to elucidate properties of alpha7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.

Rat neuronal nicotinic acetylcholine receptors containing α7 subunit: pharmacological properties of ligand binding and function

To compare pharmacological properties of heterologously expressed homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) with those of native nAChRs containing alpha7 subunit (alpha7* nAChRs) in rat hippocampus and cerebral cortex. We established a stably transfected HEK-293 cell line that expresses homomeric rat alpha7 nAChRs. We studies ligand binding profiles and functional properties of nAChRs expressed in this cell line and native rat alpha7* nAChRs in rat hippocampus and cerebral cortex. We used [(125)I]-alpha-bungarotoxin to compare ligand binding profiles in these cells with those in rat hippocampus and cerebral cortex. The functional properties of the alpha7 nAChRs expressed in this cell line were studied using whole-cell current recording. The newly established cell line, KXalpha7R1, expresses homomeric alpha7 nAChRs that bind [(125)I]-alpha-bungarotoxin with a K(d) value of 0.38+/-0.06 nmol/L, similar to K(d) values of native rat alpha7* nAChRs from hippocampus (K(d)=0.28+/-0.03 nmol/L) and cerebral cortex (K(d)=0.33+/-0.05 nmol/L). Using whole-cell current recording, the homomeric alpha7 nAChRs expressed in the cells were activated by acetylcholine and (-)-nicotine with EC(50) values of 280+/-19 micromol/L and 180+/-40 micromol/L, respectively. The acetylcholine activated currents were potently blocked by two selective antagonists of alpha7 nAChRs, alpha-bungarotoxin (IC(50)=19+/-2 nmol/L) and methyllycaconitine (IC(50)=100+/-10 pmol/L). A comparative study of ligand binding profiles, using 13 nicotinic ligands, showed many similarities between the homomeric alpha7 nAChRs and native alpha7* receptors in rat brain, but it also revealed several notable differences. This newly established stable cell line should be very useful for studying the properties of homomeric alpha7 nAChRs and comparing these properties to native alpha7* nAChRs.

Nicotinic acetylcholine receptor-mediated calcium signaling in the nervous system

Based on the composition of the five subunits forming functional neuronal nicotinic acetylcholine receptors (nAChRs), they are grouped into either heteromeric (comprising both alpha and beta subunits) or homomeric (comprising only alpha subunits) receptors. The nAChRs are known to be differentially permeable to calcium ions, with the alpha7 nAChR subtype having one of the highest permeabilities to calcium. Calcium influx through nAChRs, particularly through the alpha-bungarotoxin-sensitive alpha7-containing nAChRs, is a very efficient way to raise cytoplasmic calcium levels. The activation of nAChRs can mediate three types of cytoplasmic calcium signals: (1) direct calcium influx through the nAChRs, (2) indirect calcium influx through voltage-dependent calcium channels (VDCCs) which are activated by the nAChR-mediated depolarization, and (3) calcium-induced calcium release (CICR) (triggered by the first two sources) from the endoplasmic reticulum (ER) through the ryanodine receptors and inositol (1,4,5)-triphosphate receptors (IP(3)Rs). Downstream signaling events mediated by nAChR-mediated calcium responses can be grouped into instantaneous effects (such as neurotransmitter release, which can occur in milliseconds after nAChR activation), short-term effects (such as the recovery of nAChR desensitization through cellular signaling cascades), and long-term effects (such as neuroprotection via gene expression). In addition, nAChR activity can be regulated by cytoplasmic calcium levels, suggesting a complex reciprocal relationship. Further advances in imaging techniques, animal models, and more potent and subtype-selective ligands for neuronal nAChRs would help in understanding the neuronal nAChR-mediated calcium signaling, and lead to the development of improved therapeutic treatments.

β2 Subunit-Containing Nicotinic Receptors Mediate Acute Nicotine-Induced Activation of Calcium/Calmodulin-Dependent Protein Kinase II-Dependent Pathways in Vivo

Nicotine is the addictive component of tobacco, and successful smoking cessation therapies must address the various processes that contribute to nicotine addiction. Thus, understanding the nicotinic acetylcholine receptor (nAChR) subtypes and subsequent molecular cascades activated after nicotine exposure is of the utmost importance in understanding the progression of nicotine dependence. One possible candidate is the calcium/calmodulin-dependent protein kinase II (CaMKII) pathway. Substrates of this kinase include the vesicle-associated protein synapsin I and the transcription factor cAMP response element-binding protein (CREB). The goal of these studies was to examine these postreceptor mechanisms after acute nicotine treatment in vivo. We first show that administration of nicotine increases CaMKII activity in the ventral tegmental area (VTA), nucleus accumbens (NAc), and amygdala. In beta2 nAChR knockout (KO) mice, nicotine does not induce an increase in kinase activity, phosphorylated (p)Synapsin I, or pCREB. In contrast, alpha7 nAChR KO mice show nicotine-induced increases in CaMKII activity and pCREB, similar to their wild-type littermates. Moreover, we show that when animals are pretreated with the CaMKII inhibitors 4-[(2S)-2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl]phenyl isoquinolinesulfonic acid ester (KN-62) and N-[2-[[[3-(4-chlorophenyl)-2 propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide (KN-93), nicotine-induced increase in the kinase activity and pCREB was attenuated in the VTA and NAc, whereas pretreatment with (2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, phosphate) (KN-92), the inactive analog, did not alter the nicotine-induced increase in pCREB. Taken together, these data suggest that the nicotine-induced increase in CaMKII activity may correlate with the nicotine-induced increase in pSynapsin I and pCREB in the VTA and NAc via beta2 subunit-containing nAChRs.

A role for Leu247 residue within transmembrane domain 2 in ginsenoside-mediated alpha7 nicotinic acetylcholine receptor regulation.

Nicotinic acetylcholine receptors (nAChRs) play important roles in nervous system functions and are involved in a variety of diseases. We previously demonstrated that ginsenosides, the active ingredients of Panax ginseng, inhibit subsets of nAChR channel currents, but not alpha7, expressed in Xenopus laevis oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of alpha7 nAChR induces alterations in channel gating properties and converts alpha7 nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(S)-ginsenoside Rg(3) (Rg(3)) activity against the alpha7 nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to Rg(3). We further characterized Rg(3) regulation of L247T receptors. We found that Rg(3) inhibition of mutant alpha7 nAChR channel currents was reversible and concentration-dependent. Rg(3) inhibition was strongly voltage-dependent and noncompetitive manner. These results indicate that the interaction between Rg(3) and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in Rg(3) interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that Rg(3) forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas Rg(3) localizes at the interface of the two wild-type receptor subunits. These results indicate that mutation of Leu247 to Thr247 induces conformational changes in the wild-type receptor and provides a binding pocket for Rg(3) at the channel pore.

Cortical α7 Nicotinic Acetylcholine Receptor and β-Amyloid Levels in Early Alzheimer Disease

To examine alpha7 nicotinic acetylcholine receptor (nAChR) binding and beta-amyloid (Abeta) peptide load in superior frontal cortex (SFC) across clinical and neuropathological stages of Alzheimer disease (AD). Quantitative measures of alpha7 nAChR by [(3)H]methyllycaconitine binding and Abeta concentration by enzyme-linked immunosorbent assay in SFC were compared across subjects with antemortem clinical classification of no cognitive impairment, mild cognitive impairment, or mild to moderate AD, and with postmortem neuropathological diagnoses. Academic medical center. Subjects Twenty-nine elderly retired clergy. Quantitative measures of alpha7 nAChR binding and Abeta peptide concentration in SFC. Higher concentrations of total Abeta peptide in SFC were associated with clinical diagnosis of mild to moderate AD (P = .02), lower Mini-Mental State Examination scores (P = .003), presence of cortical Abeta plaques (P = .02), and likelihood of AD diagnosis by the National Institute on Aging-Reagan criteria (P = .002). Increased alpha7 nAChR binding was associated with National Institute on Aging-Reagan diagnosis (P = .02) and, albeit weakly, the presence of cortical Abeta plaques (P = .08). There was no correlation between the 2 biochemical measures. These observations suggest that during the clinical progression from normal cognition to neurodegenerative disease state, total Abeta peptide concentration increases while alpha7 nAChRs remain relatively stable in SFC. Regardless of subjects' clinical status, however, elevated alpha7 nAChR binding is associated with increased Abeta plaque pathology, supporting the hypothesis that cellular expression of these receptors may be upregulated selectively in Abeta plaque-burdened brain areas.

Possible Drug Candidates for Alzheimers Disease Deduced from Studying their Binding Interactions with α7 Nicotinic Acetylcholine Receptor

Dysfunction in alpha7 nicotinic acetylcholine receptor (nAChR), a member of the Cys-loop ligand-gated ion channel superfamily, is responsible for attentional and cognitive deficits in Alzheimer's disease (AD). To provide useful information for finding drug candidates for the treatment of AD, a study was carried out according to the following procedures. (1) DMXBA, a partial agonist of the alpha7 nAChR, was used as a template molecule. (2) To reduce the number of compounds to be considered, the similarity search and flexible alignment were conducted to exclude those molecules which did not match the template. (3) The molecules thus obtained were docked to alpha7 nAChR. (4) To gain more structural information, the molecular dynamics (MD) simulations were carried out for 9 most favorable agonists obtained by the aforementioned docking studies. (5) By analyzing the hydrogen bond interaction and hydrophobic/hydrophilic interaction, the following seven compounds were singled out as possible drug candidates for AD therapy: gx-50, gx-51, gx-52, gx-180, open3d-99008, open3d-51265, open3d-60247.

Expression of SLURP‐1, an endogenous α7 nicotinic acetylcholine receptor allosteric ligand, in murine bronchial epithelial cells

Mammalian secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) is a positive allosteric ligand for alpha7 nicotinic acetylcholine (ACh) receptors (alpha7 nAChRs) that potentiates responses to ACh and elicits proapoptotic activity in human keratinocytes. Mutations in the gene encoding SLURP-1 have been detected in patients with Mal de Meleda, a rare autosomal recessive skin disorder characterized by transgressive palmoplantar keratoderma. On the basis of these findings, SLURP-1 is postulated to be involved in regulating tumor necrosis factor-alpha (TNF-alpha) release from keratinocytes and macrophages via alpha7 nAChR-mediated pathways. In the present study, we assessed SLURP-1 expression in lung tissue from C57BL/6J mice to investigate the functions of SLURP-1 in pulmonary physiology and pathology. Immunohistochemical and in situ hybridization analyses revealed expression of SLURP-1 protein and mRNA, respectively, exclusively in ciliated bronchial epithelial cells. This was supported by Western blotting showing the presence of the 9.5-kDa SLURP-1 protein in whole-lung tissue and trachea. In addition, high-affinity choline transporter (CHT1) was detected in apical regions of bronchial epithelial cells and in neurons located in the lamina propria of the bronchus, suggesting that bronchial epithelial cells are able to synthesize both SLURP-1 and ACh. We also observed direct contact between F4/80-positive macrophages and bronchial epithelial cells and the presence of invading macrophages in close proximity to CHT1-positive nerve elements. Collectively, these results suggest that SLURP-1 contributes to the maintenance of bronchial epithelial cell homeostasis and to the regulation of TNF-alpha release from macrophages in bronchial tissue.

Presence of α7 nicotinic acetylcholine receptors on dorsal root ganglion neurons proved using knockout mice and selective α‐neurotoxins in histochemistry

In complex tissues where multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed, immunohistochemistry has been the most popular tool for investigation of nAChR subunit distribution. However, recent studies with nAChR subunit knockout mice demonstrated that a large panel of antibodies is unsuitable. Thus, we aimed to develop a histochemical method for selective labeling of alpha7 nAChR with neurotoxins, utilizing alpha7 nAChR-transfected cells, dorsal root ganglia (DRG) and spinal cord from wild-type and knockout mouse. The specificity of Alexa Fluor 488-conjugated alpha-bungarotoxin (Alexa-alphaBgt) was demonstrated in binding to alpha7-transfected cells inhibited by long-chain alpha-cobratoxin (CTX), but not short-chain alpha-neurotoxin II (NTII). In contrast, binding to Torpedo muscle-type nAChRs and to motor end plates in mouse tongue sections was prevented by both CTX and NTII. In tissue sections of DRG, expressing all neuronal nAChR subunits, only CTX precluded Alexa-alphaBgt labeling of neurons, with no staining for alpha7 nAChR knockout tissue. It proved that alpha7 nAChRs are the major alphaBgt-binding sites in mouse DRG. Corresponding results were obtained for terminals in the spinal cord. Thus, we present a protocol utilizing Alexa-alphaBgt and non-labeled CTX/NTII that allows specific histochemical detection of alpha7 nAChR with a spatial resolution at the level of single axon terminals.