Α4 nAChR Subunits Research Articles

Nicotine induction of theta frequency oscillations in rodent medial septal diagonal band in vitro

This study aimed to examine the role of the nicotinic receptor (nAChR) in the generation of theta oscillations (4-12 Hz) in vitro. Electrophysiological studies were performed on medial septal diagonal band area (MSDB) slices to measure theta oscillation. Immunofluorescence and confocal microscopy studies were carried out to detect α4 nAChR and β2 nAChR subunits in perfused-fixed tissue from VGluT2-GFP and GAD67-GFP transgenic mice. Application of nicotine to MSDB slices produced persistent theta oscillations in which area power increased in a dose-responsive manner. This activity was inhibited by GABAA receptor antagonists and partially by ionotropic glutamate receptor antagonists, indicating the involvement of local GABAergic and glutamatergic neurons in the production of the rhythmic activity. The nicotine-induced theta activity was also inhibited selectively by non-α7*nAChR antagonists, suggesting the presence of these receptor types on GABAergic and glutamatergic neuron populations in the MSDB. This was confirmed by immunofluorescence and confocal microscopy studies in transgenic mice in which the GABAergic and glutamatergic neurons express green fluorescent protein (GFP), showing localisation of β2 nAChR and α4 nAChR subunits, the most common constituents of non-α7*nAChRs, in both cell types in the MSDB. Theta activity in the MSDB may be generated by tonic stimulation of non-α7*nAChRs.

Regulation of glutamate release by heteromeric nicotinic receptors in layer V of the secondary motor region (Fr2) in the dorsomedial shoulder of prefrontal cortex in mouse

We studied how nicotinic acetylcholine receptors (nAChRs) regulate glutamate release in the secondary motor area (Fr2) of the dorsomedial murine prefrontal cortex, in the presence of steady agonist levels. Fr2 mediates response to behavioral situations that require immediate attention and is a candidate for generating seizures in the frontal epilepsies caused by mutant nAChRs. Morphological analysis showed a peculiar chemoarchitecture and laminar distribution of pyramidal cells and interneurons. Tonic application of 5 µM nicotine on Layer V pyramidal neurons strongly increased the frequency of spontaneous glutamatergic excitatory postsynaptic currents. The effect was inhibited by 1 µM dihydro-β-erythroidine (which blocks α4-containing nAChRs) but not by 10 nM methyllicaconitine (which blocks α7-containing receptors). Excitatory postsynaptic currents s were also stimulated by 5-iodo-3-[2(S)-azetidinylmethoxy]pyridine, selective for β2-containing receptors, in a dihydro-β-erythroidine -sensitive way. We next studied the association of α4 with different populations of glutamatergic terminals, by using as markers the vesicular glutamate transporter type (VGLUT) 1 for corticocortical synapses and VGLUT2 for thalamocortical projecting fibers. Immunoblots showed higher expression of α4 in Fr2, as compared with the somatosensory cortex. Immunofluorescence showed intense VGLUT1 staining throughout the cortical layers, whereas VGLUT2 immunoreactivity displayed a more distinct laminar distribution. In Layer V, colocalization of α4 nAChR subunit with both VGLUT1 and VGLUT2 was considerably stronger in Fr2 than in somatosensory cortex. Thus, in Fr2, α4β2 nAChRs are expressed in both intrinsic and extrinsic glutamatergic terminals and give a major contribution to control glutamate release in Layer V, in the presence of tonic agonist levels.

Preventing Expression of the Nicotinic Receptor Subunit α7 in SH-SY5Y Cells with Interference RNA Indicates that this Receptor may Protect Against the Neurotoxicity of Aβ

The present aim was to characterize the influence of the α7 nicotinic acetylcholine receptor (nAChR) on BACE, the enzyme that cleaves the amyloid precursor protein (APP) at the β-site, as well as on the oxidative stress induced by amyloid-β peptide (Aβ). To this end, human neuroblastoma SH-SY5Y cells were transfected with siRNAs targeting the α7 nAChR subunit and/or exposed to Aβ1-42. For α7 nAChR, BACE1 (cleaving at the β-site of APP) and BACE2 (cleaving within the Aβ domain), α-secretase (ADAM10), and the two components of γ-secretase, PS and NCT, the mRNA and protein levels were determined by real-time PCR and Western blotting, respectively. The level of Aβ1-42 in the cell culture medium was determined by an ELISA procedure. The extent of lipid peroxidation and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were assayed spectrophotometrically. In the transfected SH-SY5Y cells, expression of α7 nAChR was reduced; the level of BACE1 increased and that of BACE2 decreased; the amount of ADAM10 lowered; and the level of PS raised. Moreover, the level of Aβ1-42 in the culture medium was elevated. Treatment of non-transfected cells with Aβ elevated the level of malondialdehyde (MDA) and lowered the activities of SOD and GSH-Px and these changes were potentiated by inhibiting expression of α7 nAChR. These results indicate that α7 nAChR plays a significant role in amyloidogenic metabolism of APP and the oxidative stress evoked by Aβ, suggesting that this receptor might help protect against the neurotoxicity of Aβ.

Nicotinic Acetylcholine Receptors are Sensors for Ethanol in Lung Fibroblasts

Chronic ethanol (EtOH) abuse in humans is known to independently increase the incidence of and mortality due to acute lung injury in at-risk individuals. However, the mechanisms by which EtOH affects lung cells remain incompletely elucidated. In earlier work, we reported that EtOH increased the expression in lung fibroblasts of fibronectin, a matrix glycoprotein implicated in lung injury and repair. This effect was blocked by α-bungarotoxin, a neurotoxin that binds certain nicotinic acetylcholine receptors (nAChRs) thereby implicating nAChRs in this process. Here, we examine the identity of these receptors. Mouse lung fibroblasts were stimulated with EtOH (60mM) or acetylcholine (100 to 500μM) and evaluated for the expression of fibronectin and nAChRs. Inhibitors to nAChRs or the antioxidant N-acetyl cysteine (NAC) were used to assess changes in fibronectin expression. Animals exposed to EtOH for up to 6weeks were used to evaluate the expression of nAChRs in vivo. First, in EtOH-treated fibroblasts, we observed increased expression of α4 and α9 nAChR subunits. Second, we found that acetylcholine, a natural ligand for nAChRs, mimicked the effects of EtOH. Dihydro-β-erythroidin hydrobromide, a competitive inhibitor of α4 nAChR, blocked the increase in fibronectin expression and cell proliferation. Furthermore, EtOH-induced fibronectin expression was inhibited in cells silenced for α4 nAChR. However, EtOH-treated cells showed increased α-bungarotoxin binding suggesting that α4 nAChR mediates the effects of EtOH via a ligand-independent pathway. Knowing there are several important cysteine residues near the ligand-binding site of α4 nAChRs, we tested the antioxidant NAC and found that it too blocked the induction of fibronectin expression by EtOH. Also, fibroblasts exposed to oxidant stress showed increased fibronectin expression that was blocked with α-bungarotoxin. Finally, we showed increased expression of α4 nAChRs in the lung tissue of mice and rats exposed to EtOH suggesting a role for these receptors in vivo. Altogether, our observations suggest that α4 nAChRs serve as sensors for EtOH-induced oxidant stress in lung fibroblasts, thereby revealing a new mechanism by which EtOH may affect lung cells and tissue remodeling and pointing to nAChRs as potential targets for intervention.

The α3β4* nicotinic acetylcholine receptor subtype mediates nicotine reward and physical nicotine withdrawal signs independently of the α5 subunit in the mouse

The 15q25 gene cluster contains genes that code for the α5, α3, and β4 nicotinic acetylcholine receptor (nAChRs) subunits, and in human genetic studies, has shown the most robust association with smoking behavior and nicotine dependence to date. The limited available animal studies implicate a role for the α5 and β4 nAChR subunits in nicotine dependence and withdrawal; however studies focusing on the behavioral role of the α3β4* nAChR receptor subtype in nicotine dependence are lacking. Because of the apparent role of the α3β4* nAChR subtype in nicotine dependence, the goal of the current study was to better evaluate the involvement of this subtype in nicotine mediated behavioral responses. Using the selective α3β4* nAChR antagonist, α-conotoxin AuIB, we assessed the role of α3β4* nAChRs in acute nicotine, nicotine reward, and physical and affective nicotine withdrawal. Because α5 has also been implicated in nicotine dependence behaviors in mice and can form functional receptors with α3β4*, we also evaluated the role of the α3β4α5* nAChR subtype in nicotine reward and somatic nicotine withdrawal signs by blocking the α3β4* nAChR subtype in α5 nAChR knockout mice with AuIB. AuIB had no significant effect on acute nicotine behaviors, but dose-dependently attenuated nicotine reward and physical withdrawal signs, with no significant effect in affective withdrawal measures. Interestingly, AuIB also attenuated nicotine reward and somatic signs in α5 nAChR knockout mice. This study shows that α3β4* nAChRs mediate nicotine reward and physical nicotine withdrawal, but not acute nicotine behaviors or affective nicotine withdrawal signs in mice. The α5 subunit is not required in the receptor assembly to mediate these effects. Our findings suggest an important role for the α3β4* nAChR subtype in nicotine reward and physical aspects of the nicotine withdrawal syndrome.

Neuroprotection of rat retinal ganglion cells mediated through alpha7 nicotinic acetylcholine receptors

Glutamate-induced excitotoxicity is thought to play an important role in several neurodegenerative diseases in the central nervous system (CNS). In this study, neuroprotection against glutamate-induced excitotoxicity was analyzed using acetylcholine (ACh), nicotine and the α7 specific nicotinic acetylcholine receptor (α7 nAChR) agonist, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), in cultured adult rat retinal neurons. Adult Long Evans rat retinas were dissociated and retinal ganglion cells (RGCs) were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured under various pharmacological conditions to demonstrate excitotoxicity and neuroprotection against excitotoxicity. After 3days, RGCs were immunostained with antibodies against the glycoprotein, Thy 1.1, counted and cell survival was assessed relative to control untreated conditions. 500μM glutamate induced excitotoxicity in large and small RGCs in an adult rat dissociated culture. After 3days in culture with glutamate, the cell survival of large RGCs decreased by an average of 48.16% while the cell survival of small RGCs decreased by an average of 42.03%. Using specific glutamate receptor agonists and antagonists, we provide evidence that the excitotoxic response was mediated through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) and N-methyl-d-aspartate (NMDA) glutamate receptors through an apoptotic mechanism. However, the excitotoxic effect of glutamate on all RGCs was eliminated if cells were cultured for an hour with 10μM ACh, 100μM nicotine or 100nM of the α7 nAChR agonist, PNU-282987, before the glutamate insult. Inhibition studies using 10nM methyllycaconitine (MLA) or α-bungarotoxin (α-Bgt) supported the hypothesis that neuroprotection against glutamate-induced excitotoxicity on rat RGCs was mediated through α7 nAChRs. In immunocytochemical studies, double-labeled experiments using antibodies against Thy 1.1 and α7 nAChR subunits demonstrated that both large and small RGCs contained α7 nAChR subunits. The data presented in this study support the hypothesis that ACh and nicotinic acetylcholine receptor (nAChR) agonists provide neuroprotection against glutamate-induced excitotoxicity in adult rat RGCs through activation of α7 nAChR subunits. These studies lay the groundwork required for analyzing the effect of specific α7 nAChR agonists using in vivo models of excitotoxicity. Understanding the type of ACh receptors involved in neuroprotection in the rat retina could ultimately lead to therapeutic treatment for any CNS disease that involves excitotoxicity.

Original article A transcript coding for a partially duplicated form of α7 nicotinic acetylcholine receptor is absent from the CD4 + T-lymphocytes of patients with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)

It has been suggested that the homomer-forming α7 subunit (CHRNA7) of the neuronal nicotinic acetylcholine receptor (nAChR) is involved in the pathogenesis of common idiopathic generalized epilepsies (IGEs), whereas mutations of the gene coding for the α4 nAChR subunit (CHRNA4) are associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Several genes encoding nAChR subunits, including a partially duplicated isoform of CHRNA7 (CHRFAM7A), are expressed in peripheral blood lymphocytes (PBLs), and are constituents of peripheral receptors corresponding to nAChRs in the brain. Moreover, a 2-bp deletion polymorphism (c.497-498delTG; rs67158670), resulting in a frame shift and truncation of the protein product of the gene, has been found in the CHRFAM7A gene and is associated with some neurological diseases. CHRFAM7A transcript levels in CD4+ T-lymphocytes were compared between ADNFLE patients harbouring the c.851C>T mutation of the CHRNA4 gene and control healthy individuals in order to determine whether there is any correlation between CHRFAM7A expression in CD4+ T-lymphocytes and the severity of epileptic symptoms. We also tested the hypothesis that the 2-bp deletion polymorphism in the partially duplicated α7 nAChR gene may be related to ADNFLE in these patients. Peripheral venous blood samples were collected from 3 individuals with ADNFLE and from 10 healthy individuals. From the isolated CD4+ T-lymphocytes, RNA was prepared and the CHRFAM7A transcript level was determined by RT-qPCR. In order to compare the CHRNA7 and CHRFAM7A sequences and genotype the -2bp polymorphism, genomic DNA was prepared from PBLs. It has been demonstrated that CHRFAM7A is expressed in CD4+ T-lymphocytes of healthy individuals, the relative abundance of the transcript being nearly equal (about 100 copies per cell), but it is not expressed in ADNFLE patients. Genotype analysis showed that the -2bp polymorphism was found in all patients as well as in seven healthy individuals. Our observations confirm the hypothesis that the CHRFAM7A gene is expressed in CD4+ T-lymphocytes of healthy individuals and that this expression is legitimate. The observed lack of CHRFAM7A expression in ADNFLE patients might be an important factor in the pathogenesis of ADNFLE.

The α5 Neuronal Nicotinic Acetylcholine Receptor Subunit Plays an Important Role in the Sedative Effects of Ethanol But Does Not Modulate Consumption in Mice

Alcohol use disorders (AUDs) are a major public health problem, and the few treatment options available to those seeking treatment offer only modest success rates. There remains a need to identify novel targets for the treatment of AUDs. The neuronal nicotinic acetylcholine receptors (nAChRs) represent a potential therapeutic target in the brain, as recent human genetic studies have implicated gene variants in the α5 nAChR subunit as high risk factors for developing alcohol dependence. Here, we evaluate the role of the α5* nAChR for ethanol (EtOH)-mediated behaviors using male α5+/+ and α5-/- transgenic mice. We characterized the effect of hypnotic doses of EtOH and investigated drinking behavior using an adapted drinking-in-the-dark (DID) paradigm that has been shown to induce high EtOH consumption in mice. We found the α5 subunit to be important in mediating the sedative effects of EtOH. The α5-/- mice showed slower recovery from EtOH-induced sleep, as measured by loss of righting reflex. Additionally, the α5-/- mice showed enhanced impairment to EtOH-induced ataxia. We found the initial sensitivity to EtOH and EtOH metabolism to be similar in both α5+/+ and α5-/- mice. Hence, the enhanced sedation is likely due to a difference in the acute tolerance of EtOH in α5-/- mice. However, the α5 subunit did not play a role in EtOH consumption for EtOH concentrations ranging from 5 to 30% using the DID paradigm. Additionally, varenicline was effective in reducing EtOH intake in α5-/- mice. Together, our data suggest that the α5 nAChR subunit is important for the sedative effects of EtOH but does not play a role in EtOH consumption in male mice. Varenicline can be a treatment option even when there is loss of function of the α5 nAChR subunit.

Molecular Mechanisms Underlying Behaviors Related to Nicotine Addiction

Tobacco smoking results in more than 5 million deaths each year and accounts for almost 90% of all deaths from lung cancer. Nicotine, the major reinforcing component of tobacco smoke, acts in the brain through the neuronal nicotinic acetylcholine receptors (nAChRs). The nAChRs are allosterically regulated, ligand-gated ion channels consisting of five membrane-spanning subunits. Twelve mammalian α subunits (α2-α10) and β subunits (β2-β4) have been cloned. The predominant nAChR subtypes in mammalian brain are those containing α4 and β2 subunits (denoted as α4β2* nAChRs). The α4β2* nAChRs mediate many behaviors related to nicotine addiction and are the primary targets for currently approved smoking cessation agents. Considering the large number of nAChR subunits in the brain, it is likely that nAChRs containing subunits in addition to α4 and β2 also play a role in tobacco smoking. Indeed, genetic variation in the CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the α5, α3, and β4 nAChR subunits, respectively, has been shown to increase vulnerability to tobacco dependence and smoking-associated diseases including lung cancer. Moreover, mice in which expression of α5 or β4 subunits has been genetically modified have profoundly altered patterns of nicotine consumption. In addition to the reinforcing properties of nicotine, the effects of nicotine on appetite, attention, and mood are also thought to contribute to establishment and maintenance of the tobacco smoking habit. Here we review recent insights into the behavioral actions of nicotine and the nAChRs subtypes involved, which likely contribute to the development of tobacco dependence in smokers.

Expression of nicotinic acetylcholine receptor subunit mRNA in mouse bladder afferent neurons

Nicotinic acetylcholine receptors (nAChR) influence bladder afferent activity and reflex sensitivity, and have been suggested as potential targets for treating detrusor overactivity. Mechanisms may include indirect effects, e.g. involving the urothelium, and direct action on nAChR expressed by afferent neurons. Here we determined the nAChR repertoire of bladder afferent neurons by retrograde neuronal tracing and laser-assisted microdissection/reverse transcriptase polymerase chain reaction (RT–PCR), and quantified retrogradely labelled nAChRα3-subunit-expressing neurons by immunohistochemistry in nAChR α3β4α5 cluster enhanced green fluorescent protein (eGFP) reporter mice. Bladder afferents distinctly expressed mRNAs encoding for nAChR-subunits α3, α6, α7, β2–4, and weakly α4. Based upon known combinatorial patterns of subunits, this predicts the expression of at least three basically different subunits of nAChR – α3∗, α6∗ and α7∗ – and of additional combinations with β-subunits and α5. Bladder afferents were of all sizes, and their majority (69%; n=1367) were eGFP–nAChRα3 positive. Immunofluorescence revealed immunoreactivities to neurofilament 68 (NF68), transient receptor potential cation channel vanilloid 1 (TRPV1), substance P (SP) and calcitonin gene-related peptide (CGRP) in eGFP–nAChRα3-positive and -negative neurons. For each antigen, all possible combinations of colocalisation with eGFP–nAChRα3 were observed, with eGFP–nAChRα3-positive bladder neurons without additional immunoreactivity being most numerous, followed by triple-labelled neurons. In conclusion, more than one population of bladder afferent neurons expresses nAChR, indicating that peripheral nicotinic initiation and modulation of bladder reflexes might result, in addition to indirect effects, from the direct activation of sensory terminals. The expression of multiple nAChR subunits offers the potential of selectively addressing functional aspects and/or sensory neuron subpopulations.

A comparative study of the effects of ABT-418 and methylphenidate on spatial memory in an animal model of ADHD

Impaired learning performance in scholastic settings is a characteristic of attention deficit hyperactivity disorder (ADHD). Our present study compares the effect of a nicotinic acetylcholine receptor (nAChR) agonist, ABT-418, and methylphenidate (MPH) on spatial memory in spontaneously hypertensive rats (SHRs), an animal model of ADHD. Neither chronic administration of ABT-418 nor MPH affected the learning performance during training in the Morris water maze. However, both compounds significantly improved memory. SHRs treated with a combination of the compounds did not perform better than either drug alone. Furthermore, the cortical α4 and β2 nAChR subunits and the hippocampal α4 subunit expression were significantly enhanced by ABT-418 treatments. Collectively, these results suggest that ABT-418 effectively improved spatial memory in an animal model of ADHD, providing a theoretical foundation for the use of a nAChR agonist in ADHD treatment.

Effects of chronic nicotine on the autocrine regulation of pancreatic cancer cells and pancreatic duct epithelial cells by stimulatory and inhibitory neurotransmitters

Pancreatic ductal adenocarcinoma (PDAC) has a mortality rate near 100%. Smoking is a documented risk factor. However, the mechanisms of smoking-associated pancreatic carcinogenesis are poorly understood. We have shown that binding of nicotine to nicotinic acetylcholine receptors (nAChRs) expressing subunits α7, α3 and α5 in PDAC and pancreatic duct epithelial cells in vitro triggered the production of the neurotransmitters noradrenaline and adrenaline by these cells. In turn, this autocrine catecholamine loop significantly stimulated cell proliferation via cyclic adenosine 3',5'-monophosphate-dependent signaling downstream of beta-adrenergic receptors. However, the observed responses only represent acute cellular reactions to single doses of nicotine whereas nicotine exposure in smokers is chronic. Using the PDAC cell lines BxPC-3 and Panc-1 and immortalized pancreatic duct epithelial cell line HPDE6-C7, our current experiments reveal a significant sensitization of the nAChR-driven autocrine catecholamine regulatory loop in cells pre-exposed to nicotine for 7 days. The resulting increase in catecholamine production was associated with significant inductions in the phosphorylation of signaling proteins ERK, CREB, Src and AKT, upregulated protein expression of nAChR subunits α3, α4, α5 and α7 and increased responsiveness to nicotine in 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide and cell migration assays. All three cell lines produced the inhibitory neurotransmitter γ-aminobutyric acid, an activity inhibited by gene knockdown of the α4β2nAChR and suppressed by chronic nicotine via receptor desensitization. All of the observed adverse effects of chronic nicotine were reversed by treatment of the cells with γ-aminobutyric acid, suggesting the potential usefulness of this agent for the improvement of PDAC intervention strategies in smokers.

α6 nAChR subunit residues that confer α‐conotoxin BuIA selectivity

Nicotinic acetylcholine receptors (nAChRs) containing α6 and/or α4 subunits modulate the release of dopamine. However, few compounds can effectively discriminate between ligand-binding sites that contain α6 vs. α4 nAChR subunits. Using a chimeric (α6/α4) subunit, we showed that α-conotoxin BuIA binds the extracellular rat α6β2 vs. α4β2 interface with ∼60,000-fold selectivity. Chimeras containing residues from the α6 subunit were inserted into the homologous position of the α4 subunit to identify critical sequence segments. The region between residues 184 and 207 in the α6 subunit accounted for the potency difference. Chimeras within this region followed by point mutations were constructed for further definition. α6 Lys185, Thr187, and Ile188 form a triad of key residues that influence BuIA binding; when these 3 α6 residues were inserted into the α4 subunit, there was an ∼2000-fold increase in toxin potency. We used a crystal structure of BuIA bound to the acetylcholine-binding protein together with the structure of the Torepedo marmorata nAChR to build a homology model of BuIA bound to the interface between α6 and β2 subunits. The results indicate that the triad of α6 residues lies outside the C loop and is distantly located from bound BuIA (>10 Å). This suggests that alterations in potency are not caused by the direct interaction between the triad and BuIA. Instead, alterations in C-loop 3-dimensional structure and/or flexibility may account for differential potency. Thr198 and Tyr205 also contributed to BuIA potency. In addition, Thr198 caused BuIA potency differences between the closely related α6 and α3 subunits. Together, the findings provide insight into differences between the α6 and other α subunits that may be exploited by α-conotoxins to achieve binding selectivity.

The Drosophila nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels

BackgroundNicotinic acetylcholine receptors (nAChRs) play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dβ1-Dβ3) and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit.ResultsA full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric ‘triplet’ nAChR (Dα5 + Dα6 + Dα7) with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7) with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in some cellular environments.ConclusionData are presented demonstrating the ability of the Drosophila Dα5 and Dα7 subunits to generate functional homomeric and also heteromeric nAChRs.

A Highly Conserved Cytoplasmic Cysteine Residue in the α4 Nicotinic Acetylcholine Receptor Is Palmitoylated and Regulates Protein Expression

Nicotinic acetylcholine receptor (nAChR) cell surface expression levels are modulated during nicotine dependence and multiple disorders of the nervous system, but the mechanisms underlying nAChR trafficking remain unclear. To determine the role of cysteine residues, including their palmitoylation, on neuronal α4 nAChR subunit maturation and cell surface trafficking, the cysteines in the two intracellular regions of the receptor were replaced with serines using site-directed mutagenesis. Palmitoylation is a post-translational modification that regulates membrane receptor trafficking and function. Metabolic labeling with [(3)H]palmitate determined that the cysteine in the cytoplasmic loop between transmembrane domains 1 and 2 (M1-M2) is palmitoylated. When this cysteine is mutated to a serine, producing a depalmitoylated α4 nAChR, total protein expression decreases, but surface expression increases compared with wild-type α4 levels, as determined by Western blotting and enzyme-linked immunoassays, respectively. The cysteines in the M3-M4 cytoplasmic loop do not appear to be palmitoylated, but replacing all of the cysteines in the loop with serines increases total and cell surface expression. When all of the intracellular cysteines in both loops are mutated to serines, there is no change in total expression, but there is an increase in surface expression. Calcium accumulation assays and high affinity binding for [(3)H]epibatidine determined that all mutants retain functional activity. Thus, our results identify a novel palmitoylation site on cysteine 273 in the M1-M2 loop of the α4 nAChR and determine that cysteines in both intracellular loops are regulatory factors in total and cell surface protein expression of the α4β2 nAChR.

Bladder outlet obstruction influences mRNA expression of cholinergic receptors on sensory neurons in mice

AimsIn patients with bladder outlet obstruction (BOO), dysregulation of bladder afferent neurons seems to contribute to irritative symptoms. Cholinergic receptors, addressed by both neuronal and non-neuronal (urothelial) acetylcholine, can alter neuronal excitability. Thus we investigated the influence of BOO on the expression of muscarinic (mAChR) and nicotinic (nAChR) acetylcholine receptors in the lumbosacral dorsal root ganglia (DRG) of mice. Main methodsBOO was induced in 13 C57/BL6 mice by partial suturing of the urethra. Eleven mice were sham-operated (loose/freely movable suture around the urethra), and eleven untreated mice served as controls. Cystometry was performed five weeks later in conscious mice. DRG at segmental levels L5-S2 were dissected and real-time quantitative PCR was performed. Expression of mAChR subtypes M1–M5 and nAChR subunits α2–7, α9–10, β2–4 was examined. Key findingsExpression of all mAChR subtypes and nAChR subunits α3–7, α10, β2–4 was detected. Expression of α2 and α9 was absent. Rank order of expression was M2>M4>M3>M5>M1, α3≥α6>α7>α4>α10>α5 and β2>β4>β3 in untreated animals. BOO mice presented distinct obstruction with development of residual urine. Sham mice showed only minimal BOO. Relative mRNA expression of nAChR subunits revealed significant reduction of α3, α5, α6, α10 and β4 in sham-operated vs. untreated mice. In BOO vs. sham-operated mice, reduction of nAChR subunits α10 (p=0.038) and α5 (p=0.053) was found. SignificanceBOO has a considerable impact on nAChR, but not on mAChR mRNA expression in sensory neurons. We hypothesize that a reduction in mRNA expression of nAChR subunits represents a link to altered sensitivity under non-obstructive conditions.

PMCA2 via PSD-95 Controls Calcium Signaling by α7-Containing Nicotinic Acetylcholine Receptors on Aspiny Interneurons

Local control of calcium concentration within neurons is critical for signaling and regulation of synaptic communication in neural circuits. How local control can be achieved in the absence of physical compartmentalization is poorly understood. Challenging examples are provided by nicotinic acetylcholine receptors that contain α7 nicotinic receptor subunits (α7-nAChRs). These receptors are highly permeable to calcium and are concentrated on aspiny dendrites of interneurons, which lack obvious physical compartments for constraining calcium diffusion. Using functional proteomics on rat brain, we show that α7-nAChRs are associated with plasma membrane calcium-ATPase pump isoform 2 (PMCA2). Analysis of α7-nAChR function in hippocampal interneurons in culture shows that PMCA2 activity limits the duration of calcium elevations produced by the receptors. Unexpectedly, PMCA2 inhibition triggers rapid calcium-dependent loss of α7-nAChR clusters. This extreme regulatory response is mediated by CaMKII, involves proteasome activity, depends on the second intracellular loop of α7-nAChR subunits, and is specific in that it does not alter two other classes of calcium-permeable ionotropic receptors on the same neurons. A critical link is provided by the scaffold protein PSD-95 (postsynaptic density-95), which is associated with α7-nAChRs and constrains their mobility as revealed by single-particle tracking on neurons. The PSD-95 link is required for PMCA2-mediated removal of α7-nAChR clusters. This three-component combination of PMCA2, PSD-95, and α7-nAChR offers a novel mechanism for tight control of calcium dynamics in neurons.

Role for α6 nicotinic receptors in l-dopa-induced dyskinesias in parkinsonian mice

l-Dopa-induced dyskinesias are a serious side effect that develops in most Parkinson's disease patients on dopamine replacement therapy. Few treatment options are available to manage dyskinesias; however, recent studies show that nicotine reduces these abnormal involuntary movements (AIMs) in parkinsonian animals by acting at nicotinic acetylcholine receptors (nAChRs). Identification of the nAChR subtypes that mediate this reduction in AIMs is important as it will help in the development of nAChR subtype selective drugs for their treatment. Here we investigate the role of α6β2* nAChRs, a subtype selectively present in the nigrostriatal pathway, using α6 nAChR subunit null mutant (α6(−/−)) mice. Wildtype and α6(−/−) mice were lesioned by unilateral injection of 6-hydroxydopamine (3 μg/μl) into the medial forebrain bundle. They were then given l-dopa (3 mg/kg) plus benserazide (15 mg/kg) 2–3 wk later. l-dopa-induced AIMs developed to a similar extent in α6(−/−) and wildtype mice. However, AIMs in α6(−/−) mice declined to ∼50% of that in wildtype mice with continued l-dopa treatment. Nicotine treatment also decreased AIMs by ∼50% in wildtype mice, although not in α6(−/−) mice. There were no effects on parkinsonism under any experimental condition. To conclude, the similar declines in l-dopa-induced AIMs in nicotine-treated wildtype mice and in α6(−/−) mice treated with and without nicotine indicate an essential role for α6β2* nAChRs in the maintenance of l-dopa-induced AIMs. These findings suggest that α6β2* nAChR drugs have potential for reducing l-dopa-induced dyskinesias in Parkinson's disease.

Nicotine exposure and the progression of chronic kidney disease: role of the α7-nicotinic acetylcholine receptor.

Clinical studies have established the role of cigarette smoking as a risk factor in the progression of chronic kidney disease (CKD). We have shown that nicotine promotes mesangial cell proliferation and hypertrophy via nonneuronal nicotinic acetylcholine receptors (nAChRs). The α7-nAChR is one of the most important subunits of the nAChRs. These studies were designed to test the hypothesis that nicotine worsens renal injury in rats with 5/6 nephrectomy (5/6Nx) and that the α7-nAChR subunit is required for these effects. We studied five different groups: Sham, 5/6Nx, 5/6Nx + nicotine (Nic; 100 μg/ml dry wt), 5/6Nx + Nic + α7-nAChR blocker methyllicaconitine (MLA; 3 mg·kg(-1)·day(-1) sq), and Sham + Nic. Blood pressure was measured by the tail-cuff method, and urine was collected for proteinuria. After 12 wk, the rats were euthanized and kidneys were collected. We observed expression of the α7-nAChR in the proximal and distal tubules. The administration of nicotine induced a small increase in blood pressure and resulted in cotinine levels similar to those found in the plasma of smokers. In 5/6Nx rats, the administration of nicotine significantly increased urinary protein excretion (onefold), worsened the glomerular injury score and increased fibronectin (∼ 50%), NADPH oxidase 4 (NOX4; ∼100%), and transforming growth factor-β expression (∼200%). The administration of nicotine to sham rats increased total proteinuria but not albuminuria, suggesting direct effects on tubular protein reabsorption. These effects were prevented by MLA, demonstrating a critical role for the α7-nAChR as a mediator of the effects of nicotine in the progression of CKD.

From men to mice: CHRNA5/CHRNA3, smoking behavior and disease.

Introduction:The nicotinic acetylcholine receptor (nAChR) gene cluster CHRNA5-A3-B4 on chromosome 15 has been the subject of a considerable body of research over recent years. Two highly correlated single nucleotide polymorphisms (SNPs) within this region—rs16969968 in CHRNA5 and rs1051730 in CHRNA3—have generated particular interest.Methods:We reviewed the literature relating to SNPs rs16969968 and rs1051730 and smoking-related phenotypes, and clinical and preclinical studies, which shed light on the mechanisms underlying these associations.Results:Following the initial discovery of an association between this locus and smoking behavior, further associations with numerous phenotypes have been subsequently identified, including smoking-related behaviors, diseases, and cognitive phenotypes. Potential mechanisms thought to underlie these have also been described, as well as possible gene × environment interaction effects.Conclusions:Perhaps counter to the usual route of scientific inquiry, these initial findings, based exclusively on human samples and strengthened by their identification through agnostic genome-wide methods, have led to preclinical research focused on determining the mechanism underlying these associations. Progress has been made using knockout mouse models, highlighting the importance of α5 nAChR subunits in regulating nicotine intake, particularly those localized to the habenula–interpeduncular nucleus pathway. Translational research seeking to evaluate the effect of nicotine challenge on brain activation as a function of rs16969968 genotype using neuroimaging technologies is now called for, which may point to new targets for novel smoking cessation therapies.