nAChR Function Research Articles

Constructing and Tuning Excitatory Cholinergic Synapses: The Multifaceted Functions of Nicotinic Acetylcholine Receptors in Drosophila Neural Development and Physiology.

Nicotinic acetylcholine receptors (nAchRs) are widely distributed within the nervous system across most animal species. Besides their well-established roles in mammalian neuromuscular junctions, studies using invertebrate models have also proven fruitful in revealing the function of nAchRs in the central nervous system. During the earlier years, both in vitro and animal studies had helped clarify the basic molecular features of the members of the Drosophila nAchR gene family and illustrated their utility as targets for insecticides. Later, increasingly sophisticated techniques have illuminated how nAchRs mediate excitatory neurotransmission in the Drosophila brain and play an integral part in neural development and synaptic plasticity, as well as cognitive processes such as learning and memory. This review is intended to provide an updated survey of Drosophila nAchR subunits, focusing on their molecular diversity and unique contributions to physiology and plasticity of the fly neural circuitry. We will also highlight promising new avenues for nAchR research that will likely contribute to better understanding of central cholinergic neurotransmission in both Drosophila and other organisms.

The nematode C. elegans senses airborne sound

SUMMARYUnlike olfaction, taste, touch, vision, and proprioception, which are widespread across animal phyla, hearing is found only in vertebrates and some arthropods. The vast majority of invertebrate species are thus considered insensitive to sound. Here, we challenge this conventional view by showing that the earless nematode C. elegans senses airborne sound at frequencies reaching the kHz range. Sound vibrates C. elegans skin, which acts as a pressure-to-displacement transducer similar to vertebrate eardrum, activates sound-sensitive FLP/PVD neurons attached to the skin, and evokes phonotaxis behavior. We identified two nAChRs that transduce sound signals independently of ACh, revealing an unexpected function of nAChRs in mechanosensation. Thus, the ability to sense airborne sound is not restricted to vertebrates and arthropods as previously thought, and might have evolved multiple times independently in the animal kingdom, suggesting convergent evolution. Our studies also demonstrate that animals without ears may not be presumed to be sound insensitive.

Design, Synthesis, and Activity of an α-Conotoxin LtIA Fluorescent Analogue.

Nicotinic acetylcholine receptors (nAChRs) are essential pentameric ligand-gated ion channels that are distributed throughout the central and peripheral nervous systems and non-neuronal tissues in mammalian species that play critical roles in a variety of neural and mental activities. The α3β2 nAChR subtype participates in pain, addiction to nicotine, and other neurophysiological and pathological activities. Owing to the lack of highly selective pharmacological tools targeting α3β2, related research on its tissue distribution and function has been hindered. α-Conotoxin (α-CTx) LtIA, discovered from Conus literatus in our lab, potently and selectively blocks α3β2 nAChR, providing an important molecular probe to study the α3β2 nAChR structure and function. We used the fluorescent molecule 5-carboxytetramethylrhodamine succinimidyl ester, which can react with the N-terminus of LtIA, to obtain a novel fluorescent analogue of LtIA (LtIA-F). The potency and selectivity of LtIA-F were tested using a two-electrode voltage clamp recording on various nAChRs expressed in Xenopus laevis oocytes. LtIA-F potently inhibited ACh-evoked currents at the α3β2 nAChR, with an IC50 value of 90.66 nM, displaying a ∼4-fold decrease in potency compared with native LtIA without a change in selectivity. The serum stability results indicated that LtIA-F exhibited stability similar to that of native LtIA. This study on an α-CTx LtIA fluorescent analogue provides a wealth of pharmacological tools to explore the structure-function relationship, distribution, and ligand binding domain of the α3β2 nAChR subtype.

Nicotinic acetylcholine receptor redux: Discovery of accessories opens therapeutic vistas

The neurotransmitter acetylcholine (ACh) acts in part through a family of nicotinic ACh receptors (nAChRs), which mediate diverse physiological processes including muscle contraction, neurotransmission, and sensory transduction. Pharmacologically, nAChRs are responsible for tobacco addiction and are targeted by medicines for hypertension and dementia. Nicotinic AChRs were the first ion channels to be isolated. Recent studies have identified molecules that control nAChR biogenesis, trafficking, and function. These nAChR accessories include protein and chemical chaperones as well as auxiliary subunits. Whereas some factors act on many nAChRs, others are receptor specific. Discovery of these regulatory mechanisms is transforming nAChR research in cells and tissues ranging from central neurons to spinal ganglia to cochlear hair cells. Nicotinic AChR-specific accessories also enable drug discovery on high-confidence targets for psychiatric, neurological, and auditory disorders.

Accessory proteins and nicotinic receptors

Neuroscience Acetylcholine was the first neurotransmitter identified, and nicotinic acetylcholine receptors (nAChRs) were the first neurotransmitter receptors isolated. Recent studies have identified a multitude of molecules and mechanisms that regulate nAChRs in different tissues. In a Review, Matta et al. discuss these discoveries and their implications for the cell biology and medicinal pharmacology of nACHRs. Many accessory factors promote the assembly and function of diverse nAChRs. Some factors are small molecules, some are proteins, some control receptor biogenesis, and some regulate channel gating. These protein chaperones and auxiliary subunits elucidate the pharmacological and physiological processes regulated by acetylcholine. Science , abg6539, this issue p. [eabg6539][1] [1]: /lookup/doi/10.1126/science.abg6539

Synthesis and Biological Evaluation of Novel Triazine Derivatives as Positive Allosteric Modulators of α7 Nicotinic Acetylcholine Receptors.

Enhancing neuronal α7 nicotinic acetylcholine receptor (α7 nAChR) function can alleviate cognitive deficits. Here, we report the design, synthesis, and evaluation of N-(4-(trifluoromethoxy)phenyl)-1,3,5-triazin-2-amine derivatives 8-10 as a series of novel α7 nAChR positive allosteric modulators (PAMs). The representative compound 10e functions as a type I PAM with an EC50 of 3.0 μM and approximately 38-fold enhancement of α7 current in the presence of agonist acetylcholine (100 μM). It specifically enhances α7 current with high selectivity. Compound 10e shows good pharmacokinetic property in mice. Intraperitoneal injection of 10e (3 mg/kg) exhibits sufficient blood-brain barrier penetration in mice. Furthermore, 10e can also rescue the auditory gating deficit in mice with schizophrenia-like behavior. Molecular docking of 10e with homopentameric α7 nAChR reveals a new mode of action. These results support the potential of 10e for treatment for schizophrenia and Alzheimer's disease.

Neurotoxic Effects of Neonicotinoids on Mammals: What Is There beyond the Activation of Nicotinic Acetylcholine Receptors?-A Systematic Review.

Neonicotinoids are a class of insecticides that exert their effect through a specific action on neuronal nicotinic acetylcholine receptors (nAChRs). The success of these insecticides is due to this mechanism of action, since they act as potent agonists of insect nAChRs, presenting low affinity for vertebrate nAChRs, which reduces potential toxic risk and increases safety for non-target species. However, although neonicotinoids are considered safe, their presence in the environment could increase the risk of exposure and toxicity. On the other hand, although neonicotinoids have low affinity for mammalian nAChRs, the large quantity, variety, and ubiquity of these receptors, combined with its diversity of functions, raises the question of what effects these insecticides can produce in non-target species. In the present systematic review, we investigate the available evidence on the biochemical and behavioral effects of neonicotinoids on the mammalian nervous system. In general, exposure to neonicotinoids at an early age alters the correct neuronal development, with decreases in neurogenesis and alterations in migration, and induces neuroinflammation. In adulthood, neonicotinoids induce neurobehavioral toxicity, these effects being associated with their modulating action on nAChRs, with consequent neurochemical alterations. These alterations include decreased expression of nAChRs, modifications in acetylcholinesterase activity, and significant changes in the function of the nigrostriatal dopaminergic system. All these effects can lead to the activation of a series of intracellular signaling pathways that generate oxidative stress, neuroinflammation and, finally, neuronal death. Neonicotinoid-induced changes in nAChR function could be responsible for most of the effects observed in the different studies.

Chronic, Episodic Nicotine Alters Hypoglossal Motor Neuron Function at a Critical Developmental Time Point in Neonatal Rats.

Developmental nicotine exposure (DNE), alters brainstem neurons that control breathing, including hypoglossal motor neurons (XIIMNs), which innervate the tongue. Here, we tested the hypothesis that chronic, episodic DNE (eDNE), which mimics nicotine replacement therapies such as e-cigarettes or nicotine gum, alters the function of nicotinic acetylcholine receptors (nAChRs), XIIMN intrinsic properties, and tongue muscle function in vivo similar to what we have observed with a chronic, sustained exposure model. We delivered nicotine to pregnant Sprague Dawley rats through drinking water and studied pups of either sex in two age groups: postnatal day (P)1–P5 and P10–P12, which encompasses a critical period in brain development. At P1–P5, eDNE was associated with delayed recovery of nAChRs from desensitization; however, there were no changes in the magnitude of desensitization, XIIMN intrinsic properties, or tongue muscle function in vivo. By P10–P12, eDNE XIIMNs had lower peak firing frequencies in response to depolarizing current injection, larger delayed rectifier potassium currents, and continued to exhibit delayed nAChR recovery. Moreover, this age group exhibited a blunted and delayed tongue muscle response to nasal occlusion in vivo, indicating that changes to XIIMN intrinsic properties is an important mechanism behind this effect, as it is not produced by altered nAChR function alone. Together, these results show that eDNE alters XIIMNs and tongue muscle function during a critical period in brain development and that the specific effects of chronic nicotine exposure may be pattern dependent.

Action of Carvacrol on Parascaris sp. and Antagonistic Effect on Nicotinic Acetylcholine Receptors.

Parascaris sp. is the only ascarid parasitic nematode in equids and one of the most threatening infectious organisms in horses. Only a limited number of compounds are available for treatment of horse helminthiasis, and Parascaris sp. worms have developed resistance to the three major anthelmintic families. In order to overcome the appearance of resistance, there is an urgent need for new therapeutic strategies. The active ingredients of herbal essential oils are potentially effective antiparasitic drugs. Carvacrol is one of the principal chemicals of essential oil from Origanum, Thymus, Coridothymus, Thymbra, Satureja and Lippia herbs. However, the antiparasitic mode of action of carvacrol is poorly understood. Here, the objective of the work was to characterize the activity of carvacrol on Parascaris sp. nicotinic acetylcholine receptor (nAChR) function both in vivo with the use of worm neuromuscular flap preparations and in vitro with two-electrode voltage-clamp electrophysiology on nAChRs expressed in Xenopus oocytes. We developed a neuromuscular contraction assay for Parascaris body flaps and obtained acetylcholine concentration-dependent contraction responses. Strikingly, we observed that 300 µM carvacrol fully and irreversibly abolished Parascaris sp. muscle contractions elicited by acetylcholine. Similarly, carvacrol antagonized acetylcholine-induced currents from both the nicotine-sensitive AChR and the morantel-sensitive AChR subtypes. Thus, we show for the first time that body muscle flap preparation is a tractable approach to investigating the pharmacology of Parascaris sp. neuromuscular system. Our results suggest an intriguing mode of action for carvacrol, being a potent antagonist of muscle nAChRs of Parascaris sp. worms, which may account for its antiparasitic potency.

Electronic Nicotine Delivery Systems (ENDS) Flavorants Act As Non‐Competitive Antagonists of nAChRs and Contribute To Reinforcement‐Related Behavior

Significance Approximately 42% of the American population who use ENDS are between the ages of 12 and 18. The dramatic rise in adolescent ENDS use is likely attributed to the continuously growing flavor market, with more than 15,000 options available. In an attempt to combat this rise in vaping, it is crucial we determine the pharmacological impact of ENDS flavorants on nicotinic acetylcholine receptors (nAChRs) in the addiction-related circuitry of the brain. Methods We utilized cultured neuroblastoma-2a cells transiently transfected to contain α4β2 and α6β2β3 nAChRs and Ca2+ influx assays to examine the pharmacology of ENDS flavorants on nAChR function. In parallel, flavorants were also examined for their impact on nAChR upregulation using confocal microscopy. Lastly, we utilized vapor self-administration assays with male and female mice to study how these flavorants alter reinforcement-related behaviors. To determine significance, we used one- and two-way ANOVAs with appropriate post-hoc analyses. Results Menthol and two green apple flavorants (farnesol and farnesene) exhibited pharmacology consistent with the designation of NCAs on α4β2 nAChRs (IC50 values of 50–100μM). Further, both menthol and farnesol induced nAChR upregulation, whereas farnesene uniquely induced a stoichiometric shift towards high-sensitivity nAChRs. In self-administration assays, we observed that male mice self-administered menthol + nicotine, green apple + nicotine, or green apple alone, and both green apple and menthol enhanced nicotine reinforcement-related behavior. However, only green apple flavorants contributed to reinforcement-related behavior in the absence of nicotine. Conclusions Although ENDS flavors have been deemed “generally recognized as safe” by the FDA, we have identified several chemical flavorants that act as NCAs of α4β2 nAChRs and exert an impact on reinforcement-related behavior in a mouse model of vaping-related behavior. Given the constant rise in flavored ENDS use, further studies on flavors, ENDS devices, and the underlying mechanisms that take place while using these products is necessary to contribute in the regulation of ENDS use.

Distinct Nicotinic Responses Map onto Electrophysiologically‐ and Morphologically‐Distinct Pyramidal Neurons Within Layer 5 of The Mouse Prefrontal Cortex

Neurotransmission mediated by acetylcholine at its nicotinic receptors (nAChRs) within the medial prefrontal cortex (mPFC) is critical for higher-order cognitive tasks. Pyramidal neurons within layer 5 of the mPFC express nAChRs and form the primary signalling outputs from this brain region. These neurons may be categorized as either regular-firing or initial burst-firing, based on threshold responses to positive current injection. The objective of this study was to use whole-cell electrophysiology and biocytin neuron labelling in young postnatal mice (age 15-20 days) of both sexes to characterize the morphology and nAChR function within each of these neuron categories. When comparing the neuron categories, we first determined that regular-firing neurons have a greater membrane resistance and spike adaptation ratio, and a lower magnitude post-firing hyperpolarization. Regular-firing neurons also exhibited a lower frequency of spontaneous excitatory postsynaptic potentials. Detailed morphological analysis determined that regular-firing neurons have more apical dendrite matter distal to the soma and more basal dendrite matter proximal to the soma. Pharmacological experiments using receptor-selective antagonists revealed that nicotinic responses are mediated by distinct nAChR isoforms that are expressed selectively on each of the two pyramidal neuron categories. Specifically, these experiments demonstrate that heteromeric α4β2* nAChRs are present on regular-firing pyramidal neurons and homomeric α7 nAChRs are present on burst-firing pyramidal neurons. Ongoing experiments aim to determine the projection targets for each type of pyramidal neuron within each sex. These novel findings suggest a detailed receptor-based mechanism by which acetylcholine neurotransmission is regulated within output neurons of mPFC layer 5, which may impact the development of cognitive circuits involving this brain region.

Functional Changes to the Modulation of α4β2* Nicotinic Acetylcholine Receptors during Postnatal Maturation

The neurotransmitter acetylcholine (ACh) acting at its α4β2* subtype of nicotinic acetylcholine receptor (nAChR) within the cerebral cortex plays an important role to support cognitive functions. ACh normally initiates a robust excitatory response at α4β2* nAChRs, although this activity may be modulated by a number of mechanisms that alter receptor function. Negative receptor modulation may occur via desensitization by the competitive agonist nicotine or via presumed negative allosteric modulation by the endogenous progesterone-derived neurosteroid 3α-hydroxy-5α-prenan-20-one (allopregnanolone; ALLO). Conversely, positive modulation may occur via positive allosteric modulation by the alkaloid galantamine. Although each form of modulation has been demonstrated to occur in α4β2* nAChRs located on pyramidal neurons within layer VI of the medial prefrontal cortex (mPFC) in young postnatal male mice, the presence and relative strength of modulation in young postnatal female mice, and at maturity for both sexes, is unclear. Using whole-cell electrophysiology in mFPC layer VI neurons, we measured the ability of nicotine (300 nM), ALLO (10 μM), and galantamine (0.1 μM) to modulate ACh activation of α4β2* nAChRs in male and female mice at postnatal day (P)15-20 (young postnatal age) and P80-120 (adulthood). For nicotine, nAChR desensitization at P15-20 was greater than at P80-120 and this result was driven by an effect of postnatal age in male mice only. In contrast, ALLO inhibited nAChR function to a similar degree at both P15-20 and P80-120. Galantamine potentiated nAChR function to a similar degree at both P15 and P80-120, although this potentiation was significantly greater in females than in males at each age. Results from this study suggest that distinct factors are involved in each type of receptor modulation, which are differentially altered during postnatal maturation and across biological sex. Future studies will focus on mechanisms by which these factors influence the modulation of α4β2* nAChRs during postnatal maturation.

Is the Antidepressant Activity of Selective Serotonin Reuptake Inhibitors Mediated by Nicotinic Acetylcholine Receptors?

It is generally assumed that selective serotonin reuptake inhibitors (SSRIs) induce antidepressant activity by inhibiting serotonin (5-HT) reuptake transporters, thus elevating synaptic 5-HT levels and, finally, ameliorates depression symptoms. New evidence indicates that SSRIs may also modulate other neurotransmitter systems by inhibiting neuronal nicotinic acetylcholine receptors (nAChRs), which are recognized as important in mood regulation. There is a clear and strong association between major depression and smoking, where depressed patients smoke twice as much as the normal population. However, SSRIs are not efficient for smoking cessation therapy. In patients with major depressive disorder, there is a lower availability of functional nAChRs, although their amount is not altered, which is possibly caused by higher endogenous ACh levels, which consequently induce nAChR desensitization. Other neurotransmitter systems have also emerged as possible targets for SSRIs. Studies on dorsal raphe nucleus serotoninergic neurons support the concept that SSRI-induced nAChR inhibition decreases the glutamatergic hyperstimulation observed in stress conditions, which compensates the excessive 5-HT overflow in these neurons and, consequently, ameliorates depression symptoms. At the molecular level, SSRIs inhibit different nAChR subtypes by noncompetitive mechanisms, including ion channel blockade and induction of receptor desensitization, whereas α9α10 nAChRs, which are peripherally expressed and not directly involved in depression, are inhibited by competitive mechanisms. According to the functional and structural results, SSRIs bind within the nAChR ion channel at high-affinity sites that are spread out between serine and valine rings. In conclusion, SSRI-induced inhibition of a variety of nAChRs expressed in different neurotransmitter systems widens the complexity by which these antidepressants may act clinically.

Conserved nicotine-activated neuroprotective pathways involve mitochondrial stress

SummaryTobacco smoking is a risk factor for several human diseases. Conversely, smoking also reduces the prevalence of Parkinson’s disease, whose hallmark is degeneration of substantia nigra dopaminergic neurons (DNs). We use C. elegans as a model to investigate whether tobacco-derived nicotine activates nicotinic acetylcholine receptors (nAChRs) to selectively protect DNs. Using this model, we demonstrate conserved functions of DN-expressed nAChRs. We find that DOP-2, a D3-receptor homolog; MCU-1, a mitochondrial calcium uniporter; PINK-1 (PTEN-induced kinase 1); and PDR-1 (Parkin) are required for nicotine-mediated protection of DNs. Together, our results support involvement of a calcium-modulated, mitochondrial stress-activated PINK1/Parkin-dependent pathway in nicotine-induced neuroprotection. This suggests that nicotine-selective protection of substantia nigra DNs is due to the confluence of two factors: first, their unique vulnerability to mitochondrial stress, which is mitigated by increased mitochondrial quality control due to PINK1 activation, and second, their specific expression of D3-receptors.

The α5 Nicotinic Acetylcholine Receptor Subunit Differentially Modulates α4β2* and α3β4* Receptors.

Nicotine, the principal reinforcing compound in tobacco, acts in the brain by activating neuronal nicotinic acetylcholine receptors (nAChRs). This review summarizes our current knowledge regarding how the α5 accessory nAChR subunit, encoded by the CHRNA5 gene, differentially modulates α4β2* and α3β4* receptors at the cellular level. Genome-wide association studies have linked a gene cluster in chromosomal region 15q25 to increased susceptibility to nicotine addiction, lung cancer, chronic obstructive pulmonary disease, and peripheral arterial disease. Interestingly, this gene cluster contains a non-synonymous single-nucleotide polymorphism (SNP) in the human CHRNA5 gene, causing an aspartic acid (D) to asparagine (N) substitution at amino acid position 398 in the α5 nAChR subunit. Although other SNPs have been associated with tobacco smoking behavior, efforts have focused predominantly on the D398 and N398 variants in the α5 subunit. In recent years, significant progress has been made toward understanding the role that the α5 nAChR subunit—and the role of the D398 and N398 variants—plays on nAChR function at the cellular level. These insights stem primarily from a wide range of experimental models, including receptors expressed heterologously in Xenopus oocytes, various cell lines, and neurons derived from human induced pluripotent stem cells (iPSCs), as well as endogenous receptors in genetically engineered mice and—more recently—rats. Despite providing a wealth of available data, however, these studies have yielded conflicting results, and our understanding of the modulatory role that the α5 subunit plays remains incomplete. Here, we review these reports and the various techniques used for expression and analysis in order to examine how the α5 subunit modulates key functions in α4β2* and α3β4* receptors, including receptor trafficking, sensitivity, efficacy, and desensitization. In addition, we highlight the strikingly different role that the α5 subunit plays in Ca2+ signaling between α4β2* and α3β4* receptors, and we discuss whether the N398 α5 subunit variant can partially replace the D398 variant.

Dual Roles of α7 Nicotinic Acetylcholine Receptors Expressed in Immune Cells in T Cell Differentiation -α7 nAChRs Exert Different Actions between Antigen-presenting Cells and CD4+ T Cells

Immune cells such as T cells, macrophages and dendritic cells express various cholinergic system components, including muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs, respectively) and choline acetyltransferase (ChAT), depending on the status of the immune system. The cholinergic system which these components comprise has important effects on the regulation of immune and inflammatory responses. α7 nAChR is a neuronal-type nAChR composed of a homopentamer of the α7 subunit and is characterized by high permeability to Ca2+. It is also expressed in immune cells. For example, α7 nAChRs expressed in B cells suppress IgG production by suppressing B cell maturation into plasma cells. In addition, α7 nAChRs expressed in macrophages suppress production and release of tumor necrosis factor (TNF)-α in a mouse lipopolysaccharide (LPS)-induced sepsis model, thereby protecting the mice from lethal shock. In this review, we summarize the functions of α7 nAChRs expressed in CD4+ helper T (Th) cells and antigen-presenting cells (APCs), such as dendritic cells and macrophages. We focus in particular on their role in Th cell differentiation. α7 nAChRs on APCs interfere with antigen presentation, which leads to suppression of Th cell differentiation. By contrast, α7 nAChRs on naïve Th cells enhance their differentiation. These distinct roles of α7 nAChRs expressed in APCs and Th cells could be useful for development of drugs and therapeutic strategies for the treatment of immune- and inflammation-related diseases and cancers.

Cholinergic Dysfunction Involvement in Chronic Cerebral Hypoperfusion-Induced Impairment of Medial Septum-dCA1 Neurocircuit in Rats.

Chronic cerebral hypoperfusion (CCH) is considered a preclinical condition of mild cognitive impairment and thought to precede dementia. However, as the principal cholinergic source of hippocampus, whether the septo-hippocampal neurocircuit was impaired after CCH is still unknown. In this study, we established the CCH rat model by bilateral common carotid artery occlusion (2VO). Under anesthesia, the medial septum (MS) of rats was stimulated to evoke the field excitatory post-synaptic potential (fEPSP) in the pyramidal cell layer of dCA1. Consequently, we observed decreased amplitude of fEPSP and increased paired-pulse ratio (PPR) after 8-week CCH. After tail pinch, we also found decreased peak frequency and shortened duration of hippocampal theta rhythm in 2VO rats, indicating the dysfunction of septo-hippocampal neurocircuit. Besides, by intracerebroventricularly injecting GABAergic inhibitor (bicuculline) and cholinergic inhibitors (scopolamine and mecamylamine), we found that CCH impaired both the pre-synaptic cholinergic release and the post-synaptic nAChR function in MS–dCA1 circuits. These results gave an insight into the role of CCH in the impairment of cholinergic MS–dCA1 neurocircuits. These findings may provide a new idea about the CCH-induced neurodegenerative changes.

Conserved Nicotine-Activated Neuroprotective Pathways Involve Mitochondrial Stress

Tobacco smoking is a risk factor for several human diseases. Conversely, smoking also reduces the prevalence of Parkinson’s disease (PD), whose hallmark is degeneration of substantia nigra dopaminergic neurons (DNs). We use C. elegans as a model to investigate whether tobacco-derived nicotine activates nicotinic acetylcholine receptors (nAChRs) to selectively protect DNs. Using this model we demonstrate conserved functions of DN-expressed nAChRs. We find that DOP-2, a D3-receptor homolog, MCU-1, a mitochondrial calcium uniporter, and PINK-1, PTEN-induced kinase 1, are required for nicotine-mediated protection of DNs. Together, our results support involvement of calcium-dependent mitochondrial stress activation of PINK-1 in nicotine-dependent neuroprotection. This suggests that nicotine’s selective protection of substantia nigra DNs is due to the confluence of two factors: first, their unique vulnerability to mitochondrial stress, which is mitigated by increased mitochondrial quality control due to PINK1 activation; and second, to their specific expression of D3 receptors.

Sequential purification and characterization of Torpedo californica nAChR-DC supplemented with CHS for high-resolution crystallization studies

Over the past 10 years we have been developing a multi-attribute analytical platform that allows for the preparation of milligram amounts of functional, high-pure, and stable Torpedo (muscle-type) nAChR detergent complexes for crystallization purpose. In the present work, we have been able to significantly improve and optimize the purity and yield of nicotinic acetylcholine receptors in detergent complexes (nAChR-DC) without compromising stability and functionality. We implemented new methods in the process, such as analysis and rapid production of samples for future crystallization preparations. Native nAChR was extracted from the electric organ of Torpedo californica using the lipid-like detergent LysoFos Choline 16 (LFC-16), followed by three consecutive steps of chromatography purification.We evaluated the effect of cholesteryl hemisuccinate (CHS) supplementation during the affinity purification steps of nAChR-LFC-16 in terms of receptor secondary structure, stability and functionality. CHS produced significant changes in the degree of β-secondary structure, these changes compromise the diffusion of the nAChR-LFC-16 in lipid cubic phase. The behavior was reversed by Methyl-β-Cyclodextrin treatment. Also, CHS decreased acetylcholine evoked currents of Xenopus leavis oocyte injected with nAChR-LFC-16 in a concentration-dependent manner. Methyl-β-Cyclodextrin treatment do not reverse functionality, however column delipidation produced a functional protein similar to nAChR-LFC-16 without CHS treatment.

The functional role of the αM4 transmembrane helix in the muscle nicotinic acetylcholine receptor probed through mutagenesis and coevolutionary analyses

The activity of the muscle-type Torpedo nicotinic acetylcholine receptor (nAChR) is highly sensitive to lipids, but the underlying mechanisms remain poorly understood. The nAChR transmembrane α-helix, M4, is positioned at the perimeter of each subunit in direct contact with lipids and likely plays a central role in lipid sensing. To gain insight into the mechanisms underlying nAChR lipid sensing, we used homology modeling, coevolutionary analyses, site-directed mutagenesis, and electrophysiology to examine the role of the α-subunit M4 (αM4) in the function of the adult muscle nAChR. Ala substitutions for most αM4 residues, including those in clusters of polar residues at both the N and C termini, and deletion of up to 11 C-terminal residues had little impact on the agonist-induced response. Even Ala substitutions for coevolved pairs of residues at the interface between αM4 and the adjacent helices, αM1 and αM3, had little effect, although some impaired nAChR expression. On the other hand, Ala substitutions for Thr422 and Arg429 caused relatively large losses of function, suggesting functional roles for these specific residues. Ala substitutions for aromatic residues at the αM4-αM1/αM3 interface generally led to gains of function, as previously reported for the prokaryotic homolog, the Erwinia chrysanthemi ligand-gated ion channel (ELIC). The functional effects of individual Ala substitutions in αM4 were found to be additive, although not in a completely independent manner. Our results provide insight into the structural features of αM4 that are important. They also suggest how lipid-dependent changes in αM4 structure ultimately modify nAChR function.