nAChR Function Research Articles

Molecular regulatory effect of the ergot alkaloid methylergometrine on the α3β4 nicotinic acetylcholine receptor.

Methylergometrine has widely been used pharmacologically to treat conditions such as pain, addiction, vasoconstriction, migraines, and Parkinson's disease. Despite its side effects, it is used as a therapeutic agent and research material for various diseases based on its natural potential; however, the regulatory effect of its interaction with the nicotinic acetylcholine receptor (nAChR) has not yet been investigated. The α3β4 nAChR is an ion channel essential for neurotransmission within the sympathetic, parasympathetic, and autonomic nervous systems. Inhibiting the α3β4 nAChR can suppress neural signaling, affecting physiological processes such as heart rate, blood pressure, and digestion. This inhibition also holds analgesic potential by blocking pain pathways. Additionally, because the α3β4 nAChR is linked to addiction, its inhibition may reduce dependence, offering promise for addiction therapies. Inhibiting the α3β4 nAChR may impact cardiovascular and gastrointestinal regulation, presenting therapeutic possibilities for related disorders. This study investigated methylergometrine's regulatory effects on the α3β4 nAChR using oocytes and a two-electrode voltage clamp. Methylergometrine inhibited α3β4 nAChR activity in a reversible, concentration-dependent, and non-competitive manner with the interaction of α3-and β4-subunit E175 and E51 residues. These findings indicate that methylergometrine modulates α3β4 nAChR function by interacting with specific ligand-binding sites, as evidenced by electrophysiological analysis. This study advances the pharmacological understanding of methylergometrine, elucidating its mechanism of action at the cellular level.

Discovery of Antinociceptive α9α10 Nicotinic Acetylcholine Receptor Antagonists by Stable Receptor Expression.

Chronic neuropathic pain is an increasingly prevalent societal issue that responds poorly to existing therapeutic strategies. The α9α10 nicotinic acetylcholine receptor (nAChR) has emerged as a potential target to treat neuropathic pain. However, challenges in expressing functional α9α10 nAChRs in mammalian cell lines have slowed the discovery of α9α10 ligands and studies into the relationship between α9α10 nAChRs and neuropathic pain. Here, we develop a cell line in the HEK293 background that stably expresses functional α9α10 nAChRs. By also developing cell lines expressing only α9 and α10 subunits, we identify distinct receptor pharmacology between homomeric α9 or α10 and heteromeric α9α10 nAChRs. Moreover, we demonstrate that incubation with nAChR ligands differentially regulates the expression of α9- or α10-containing nAChRs, suggesting a possible mechanism by which ligands may modify receptor composition and trafficking in α9- and α10-expressing cells. We then apply our α9α10 cell line in a screen of FDA-approved and investigational drugs to identify α9α10 ligands that provide new tools to probe α9α10 nAChR function. We demonstrate that one compound from this screen, diphenidol, possesses antinociceptive activity in a murine model of neuropathic pain. These results expand our understanding of α9α10 receptor pharmacology and provide new starting points for developing efficacious neuropathic pain treatments.

Alterations in Neuronal Nicotinic Acetylcholine Receptors in the Pathogenesis of Various Cognitive Impairments.

Cognitive impairment is a typical symptom of both neurodegenerative and certain other diseases. In connection with these different pathologies, the etiology and neurological and metabolic changes associated with cognitive impairment must differ. Until these characteristics and differences are understood in greater detail, pharmacological treatment of the different forms of cognitive impairment remains suboptimal. Neurotransmitter receptors, including neuronal nicotinic acetylcholine receptors (nAChRs), dopamine receptors, and glutamine receptors, play key roles in the functions and metabolisms of the brain. Among these, the role of nAChRs in the development of cognitive impairment has attracted more and more attention. The present review summarizes what is presently known concerning the structure, distribution, metabolism, and function of nAChRs, as well as their involvement in major cognitive disorders such as Alzheimer's disease, Parkinson's disease, vascular dementia, schizophrenia, and diabetes mellitus. As will be discussed, the relevant scientific literature reveals clearly that the α4β2 and α7 nAChR subtypes and/or subunits of the receptors play major roles in maintaining cognitive function and in neuroprotection of the brain. Accordingly, focusing on these as targets of drug therapy can be expected to lead to breakthroughs in the treatment of cognitive disorders such as AD and schizophrenia.

The Smoky Impact of Nicotinic Acetylcholine Receptors on Testicular Function.

Smoking habits (from classic cigarettes to e-cigarettes and heated tobacco) are a relatively common finding in the medical histories of couples referred to fertility centers. Tobacco smoke and e-cigarettes may deliver many substances with known harmful effects on both general and reproductive health, including nicotine. Nicotinic Acetylcholine receptors (nAChRs) form a heterogeneous family of ion channels that are differently expressed in different tissues. According to the homomeric or heteromeric combination of at least five different subunits (named from α to ε), they have peculiar pharmacological and biophysical properties. nAChRs respond to the neurotransmitter acetylcholine, which influences a number of physiological functions not restricted to neurons and plays an important role in the structure and function of non-neuronal tissues such as the testis. nAChRs are also the target of Nicotine, the active element responsible for tobacco addiction. This review summarizes recent findings on the involvement of nAChRs in testicular physiology, highlighting the effects of nicotine exposure observed in animal studies and clinical settings. We will discuss the latest data on fertility outcomes and the implications for understanding nAChR functions in reproductive health.

Targeting high-affinity nicotinic receptors protects against the functional consequences of β-amyloid in mouse hippocampus.

The accumulation of β-amyloid oligomers is a hallmark of Alzheimer's disease, inducing neural and network dysfunction in the early stages of pathology. The hippocampus is affected early in the pathogenesis of AD, however the impact of soluble β-amyloid on the dentate gyrus (DG) subregion of the hippocampus and its interaction with nicotinic acetylcholine receptors (nAChRs) within this region are not known. Using a localized model of over-expression, we show that β-amyloid induces early-onset neuronal hyperactivity and hippocampal-dependent memory deficits in mice. Further, we find the DG region to be under potent and sub-type specific nicotinic control in both healthy and pathophysiological conditions, with targeted receptor inhibition leading to a mnemonic rescue against localized amyloidosis. We show that while neurogenesis and synaptic functions are not severely affected in our model, reducing β2-containing nAChR function is associated with the promotion of young adult-born neurons within the pathological network, suggesting a possible protective mechanism. Our data thus reveal the DG network level changes which occur in the early-stages of β-amyloid accumulation and highlight the downstream consequences of targeted nicotinic neuromodulation.

N-Glycosylation Deficiency in Transgene α7 nAChR and RIC3 Expressing CHO Cells Without NACHO.

The human neuronal nicotinic acetylcholine receptor α7 (nAChR) is an important target implicated in diseases like Alzheimer's or Parkinson's, as well as a validated target for drug discovery. For α7 nAChR model systems, correct folding and ion influx functions are essential. Two chaperones, resistance to inhibitors of cholinesterase 3 (RIC3) and novel nAChR regulator (NACHO), enhance the assembly and function of α7 nAChR. This study investigates the consequence of NACHO absence on α7 nAChR expression and function. Therefore, the sequences of human α7 nAChR and human RIC3 were transduced in Chinese hamster ovary (CHO) cells. Protein expression and function of α7 nAChR were confirmed by Western blot and voltage clamp, respectively. Cellular viability was assessed by cell proliferation and lactate dehydrogenase assays. Intracellular and extracellular expression were determined by in/on-cell Western, compared with another nAChR subtype by novel cluster fluorescence-linked immunosorbent assay, and N-glycosylation efficiency was assessed by glycosylation digest. The transgene CHO cell line showed expected protein expression and function for α7 nAChR and cell viability was barely influenced by overexpression. While intracellular levels of α7 nAChR were as anticipated, plasma membrane insertion was low. The glycosylation digest revealed no appreciable N-glycosylation product. This study demonstrates a stable and functional cell line expressing α7 nAChR, whose protein expression, function, and viability are not affected by the absence of NACHO. The reduced plasma membrane insertion of α7 nAChR, combined with incorrect matured N-glycosylation at the Golgi apparatus, suggests a loss of recognition signal for lectin sorting.

Fluorescent α-Conotoxin [Q1G, ΔR14]LvIB Identifies the Distribution of α7 Nicotinic Acetylcholine Receptor in the Rat Brain.

α7 nicotinic acetylcholine receptors (nAChRs) are mainly distributed in the central nervous system (CNS), including the hippocampus, striatum, and cortex of the brain. The α7 nAChR has high Ca2+ permeability and can be quickly activated and desensitized, and is closely related to Alzheimer's disease (AD), epilepsy, schizophrenia, lung cancer, Parkinson's disease (PD), inflammation, and other diseases. α-conotoxins from marine cone snail venom are typically short, disulfide-rich neuropeptides targeting nAChRs and can distinguish various subtypes, providing vital pharmacological tools for the functional research of nAChRs. [Q1G, ΔR14]LvΙB is a rat α7 nAChRs selective antagonist, modified from α-conotoxin LvΙB. In this study, we utilized three types of fluorescein after N-Hydroxy succinimide (NHS) activation treatment: 6-TAMRA-SE, Cy3 NHS, and BODIPY-FL NHS, labeling the N-Terminal of [Q1G, ΔR14]LvΙB under weak alkaline conditions, obtaining three fluorescent analogs: LvIB-R, LvIB-C, and LvIB-B, respectively. The potency of [Q1G, ΔR14]LvΙB fluorescent analogs was evaluated at rat α7 nAChRs expressed in Xenopus laevis oocytes. Using a two-electrode voltage clamp (TEVC), the half-maximal inhibitory concentration (IC50) values of LvIB-R, LvIB-C, and LvIB-B were 643.3 nM, 298.0 nM, and 186.9 nM, respectively. The stability of cerebrospinal fluid analysis showed that after incubation for 12 h, the retention rates of the three fluorescent analogs were 52.2%, 22.1%, and 0%, respectively. [Q1G, ΔR14]LvΙB fluorescent analogs were applied to explore the distribution of α7 nAChRs in the hippocampus and striatum of rat brain tissue and it was found that Cy3- and BODIPY FL-labeled [Q1G, ΔR14]LvΙB exhibited better imaging characteristics than 6-TAMARA-. It was also found that α7 nAChRs are widely distributed in the cerebral cortex and cerebellar lobules. Taking into account potency, imaging, and stability, [Q1G, ΔR14]LvΙB -BODIPY FL is an ideal pharmacological tool to investigate the tissue distribution and function of α7 nAChRs. Our findings not only provide a foundation for the development of conotoxins as visual pharmacological probes, but also demonstrate the distribution of α7 nAChRs in the rat brain.

Human restricted CHRFAM7A gene increases brain efficiency.

CHRFAM7A, a uniquely human fusion gene, has been associated with neuropsychiatric disorders including Alzheimer's disease, schizophrenia, anxiety, and attention deficit disorder. Understanding the physiological function of CHRFAM7A in the human brain is the first step to uncovering its role in disease. CHRFAM7A was identified as a potent modulator of intracellular calcium and an upstream regulator of Rac1 leading to actin cytoskeleton reorganization and a switch from filopodia to lamellipodia implicating a more efficient neuronal structure. We performed a neurocognitive-MRI correlation exploratory study on 46 normal human subjects to explore the effect of CHRFAM7A on human brain. Dual locus specific genotyping of CHRFAM7A was performed on genomic DNA to determine copy number (TaqMan assay) and orientation (capillary sequencing) of the CHRFAM7A alleles. As only the direct allele is expressed at the protein level and affects α7 nAChR function, direct allele carriers and non-carriers are compared for neuropsychological and MRI measures. Subjects underwent neuropsychological testing to measure motor (Timed 25-foot walk test, 9-hole peg test), cognitive processing speed (Symbol Digit Modalities Test), Learning and memory (California Verbal Learning Test immediate and delayed recall, Brief Visuospatial Memory Test-Revised immediate and delayed recall) and Beck Depression Inventory-Fast Screen, Fatigue Severity Scale. All subjects underwent MRI scanning on the same 3 T GE scanner using the same protocol. Global and tissue-specific volumes were determined using validated cross-sectional algorithms including FSL's Structural Image Evaluation, using Normalization, of Atrophy (SIENAX) and FSL's Integrated Registration and Segmentation Tool (FIRST) on lesion-inpainted images. The cognitive tests were age and years of education-adjusted using analysis of covariance (ANCOVA). Age-adjusted analysis of covariance (ANCOVA) was performed on the MRI data. CHRFAM7A direct allele carrier and non-carrier groups included 33 and 13 individuals, respectively. Demographic variables (age and years of education) were comparable. CHRFAM7A direct allele carriers demonstrated an upward shift in cognitive performance including cognitive processing speed, learning and memory, reaching statistical significance in visual immediate recall (FDR corrected p = 0.018). The shift in cognitive performance was associated with smaller whole brain volume (uncorrected p = 0.046) and lower connectivity by resting state functional MRI in the visual network (FDR corrected p = 0.027) accentuating the cognitive findings. These data suggest that direct allele carriers harbor a more efficient brain consistent with the cellular biology of actin cytoskeleton and synaptic gain of function. Further larger human studies of cognitive measures correlated with MRI and functional imaging are needed to decipher the impact of CHRFAM7A on brain function.

State-dependent binding of cholesterol and an anionic lipid to the muscle-type Torpedo nicotinic acetylcholine receptor

The ability of the Torpedo nicotinic acetylcholine receptor (nAChR) to undergo agonist-induced conformational transitions requires the presence of cholesterol and/or anionic lipids. Here we use recently solved structures along with multiscale molecular dynamics simulations to examine lipid binding to the nAChR in bilayers that have defined effects on nAChR function. We examine how phosphatidic acid and cholesterol, lipids that support conformational transitions, individually compete for binding with phosphatidylcholine, a lipid that does not. We also examine how the two lipids work synergistically to stabilize an agonist-responsive nAChR. We identify rapidly exchanging lipid binding sites, including both phospholipid sites with a high affinity for phosphatidic acid and promiscuous cholesterol binding sites in the grooves between adjacent transmembrane α-helices. A high affinity cholesterol site is confirmed in the inner leaflet framed by a key tryptophan residue on the MX α-helix. Our data provide insight into the dynamic nature of lipid-nAChR interactions and set the stage for a detailed understanding of the mechanisms by which lipids facilitate nAChR function at the neuromuscular junction.

CHRFAM7A diversifies human immune adaption through Ca2+ signalling and actin cytoskeleton reorganization

Human restricted genes contribute to human specific traits in the immune system. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of the α7 nicotinic acetylcholine receptor (α7 nAChR), the highest Ca2+ conductor of the ACh receptors implicated in innate immunity. Understanding the mechanism of how CHRFAM7A affects the immune system remains unexplored. Two model systems are used, human induced pluripotent stem cells (iPSC) and human primary monocytes, to characterize α7 nAChR function, Ca2+ dynamics and decoders to elucidate the pathway from receptor to phenotype. CHRFAM7A/α7 nAChR is identified as a hypomorphic receptor with mitigated Ca2+ influx and prolonged channel closed state. This shifts the Ca2+ reservoir from the extracellular space to the endoplasmic reticulum (ER) leading to Ca2+ dynamic changes. Ca2+ decoder small GTPase Rac1 is then activated, reorganizing the actin cytoskeleton. Observed actin mediated phenotypes include cellular adhesion, motility, phagocytosis and tissue mechanosensation. CHRFAM7A introduces an additional, human specific, layer to Ca2+ regulation leading to an innate immune gain of function. Through the actin cytoskeleton it drives adaptation to the mechanical properties of the tissue environment leading to an ability to invade previously immune restricted niches. Human genetic diversity predicts profound translational significance as its understanding builds the foundation for successful treatments for infectious diseases, sepsis, and cancer metastasis. This work is supported in part by the Community Foundation for Greater Buffalo (Kinga Szigeti) and in part by NIH grant R01HL163168 (Yongho Bae).

Allosteric Activation of α7 Nicotinic Acetylcholine Receptors by Novel 2-Arylamino-thiazole-5-carboxylic Acid Amide Derivatives for the Improvement of Cognitive Deficits in Mice.

Enhancing α7 nAChR function serves as a therapeutic strategy for cognitive disorders. Here, we report the synthesis and evaluation of 2-arylamino-thiazole-5-carboxylic acid amide derivatives 6-9 that as positive allosteric modulators (PAMs) activate human α7 nAChR current expressed in Xenopus ooctyes. Among the 4-amino derivatives, a representative atypical type I PAM 6p exhibits potent activation of α7 current with an EC50 of 1.3 μM and the maximum activation effect on the current over 48-fold in the presence of acetylcholine (100 μM). The structure-activity relationship (SAR) analysis reveals that the 4-amino group is crucial for the allosteric activation of α7 currents by compound 6p as the substitution of 4-methyl group results in its conversion to compound 7b (EC50 = 2.1 μM; max effect: 58-fold) characterized as a typical type I PAM. Furthermore, both 6p and 7b are able to rescue auditory gating deficits in mouse schizophrenia-like model of acoustic startle prepulse inhibition.

Subtype-Selective Peptide and Protein Neurotoxic Inhibitors of Nicotinic Acetylcholine Receptors Enhance Proliferation of Patient-Derived Glioblastoma Cell Lines.

Glioblastoma multiforme (GBM) is the most aggressive type of brain cancer, with a poor prognosis. GBM cells, which develop in the environment of neural tissue, often exploit neurotransmitters and their receptors to promote their own growth and invasion. Nicotinic acetylcholine receptors (nAChRs), which play a crucial role in central nervous system signal transmission, are widely represented in the brain, and GBM cells express several subtypes of nAChRs that are suggested to transmit signals from neurons, promoting tumor invasion and growth. Analysis of published GBM transcriptomes revealed spatial heterogeneity in nAChR subtype expression, and functional nAChRs of α1*, α7, and α9 subtypes are demonstrated in our work on several patient-derived GBM microsphere cultures and on the U87MG GBM cell line using subtype-selective neurotoxins and fluorescent calcium mobilization assay. The U87MG cell line shows reactions to nicotinic agonists similar to those of GBM patient-derived culture. Selective α1*, α7, and α9 nAChR neurotoxins stimulated cell growth in the presence of nicotinic agonists. Several cultivating conditions with varying growth factor content have been proposed and tested. The use of selective neurotoxins confirmed that cell cultures obtained from patients are representative GBM models, but the use of media containing fetal bovine serum can lead to alterations in nAChR expression and functioning.

BNC210, a negative allosteric modulator of the alpha 7 nicotinic acetylcholine receptor, demonstrates anxiolytic- and antidepressant-like effects in rodents

This work describes the characterization of BNC210 (6-[(2,3-dihydro-1H-inden-2-yl)amino]-1-ethyl-3-(4-morpholinylcarbonyl)-1,8-naphthyridin-4(1H)-one), a selective, small molecule, negative allosteric modulator (NAM) of α7 nicotinic acetylcholine receptors (α7 nAChR). With the aim to discover a non-sedating, anxiolytic compound, BNC210 was identified during phenotypic screening of a focused medicinal chemistry library using the mouse Light Dark (LD) box to evaluate anxiolytic-like activity and the mouse Open Field (OF) (dark) test to detect sedative and/or motor effects. BNC210 exhibited anxiolytic-like activity with no measurable sedative or motor effects. Electrophysiology showed that BNC210 did not induce α7 nAChR currents by itself but inhibited EC80 agonist-evoked currents in recombinant GH4C1 cell lines stably expressing the rat or human α7 nAChR. BNC210 was not active when tested on cell lines expressing other members of the cys-loop ligand-gated ion channel family. Screening over 400 other targets did not reveal any activity for BNC210 confirming its selectivity for α7 nAChR. Oral administration of BNC210 to male mice and rats in several tests of behavior related to anxiety- and stress- related disorders, demonstrated significant reduction of these behaviors over a broad therapeutic range up to 500 times the minimum effective dose. Further testing for potential adverse effects in suitable rat and mouse tests showed that BNC210 did not produce sedation, memory and motor impairment or physical dependence, symptoms associated with current anxiolytic therapeutics. These data suggest that allosteric inhibition of α7 nAChR function may represent a differentiated approach to treating anxiety- and stress- related disorders with an improved safety profile compared to current treatments.

Recombinant cellular model system for human muscle-type nicotinic acetylcholine receptor α12β1δε

The human muscle-type nicotinic acetylcholine receptor α12β1δε (nAChR) is a complex transmembrane receptor needed for drug screening for disorders like congenital myasthenic syndromes and multiple pterygium syndrome. Until today, most models are still using the nAChR from Torpedo californica electric ray. A simple reproducible cellular system expressing functional human muscle-type nAChR is still missing. This study addressed this issue and further tested the hypothesis that different chaperones, both biological and chemical, and posttranslational modification supporting substances as well as hypothermic incubation are able to increase the nAChR yield. Therefore, Gibson cloning was used to generate transfer plasmids carrying the sequence of nAChR or chosen biological chaperones to support the nAChR folding in the cellular host. Viral transduction was used for stable integration of these transgenes in Chinese hamster ovary cells (CHO). Proteins were detected with Western blot, in-cell and on-cell Western, and the function of the receptor with voltage clamp analysis. We show that the internalization of nAChR into plasma membranes was sufficient for detection and function. Additional transgenic overexpression of biological chaperones did result in a reduced nAChR expression. Chemical chaperones, posttranslational modification supporting substances, and hypothermic conditions are well-suited supporting applications to increase the protein levels of different subunits. This study presents a stable and functional cell line that expresses human muscle-type nAChR and yields can be further increased using the chemical chaperone nicotine without affecting cell viability. The simplified access to this model system should enable numerous applications beyond drug development.Graphical abstractGraphical abstract created with http://biorender.com

Three finger toxins of elapids: structure, function, clinical applications and its inhibitors.

The WHO lists snakebite as a "neglected tropical disease". In tropical and subtropical areas, envenoming is an important public health issue. This review article describes the structure, function, chemical composition, natural inhibitors, and clinical applications of Elapids' Three Finger Toxins (3FTX) using scientific research data. The primary venomous substance belonging to Elapidae is 3FTX, that targets nAChR. Three parallel β-sheets combine to create 3FTX, which has four or five disulfide bonds. The three primary types of 3FTX are short-chain, long-chain, and nonconventional 3FTX. The functions of 3FTX depend on the specific toxin subtype and the target receptor or ion channel. The well-known effect of 3FTX is probably neurotoxicity because of the severe consequences of muscular paralysis and respiratory failure in snakebite victims. 3FTX have also been studied for their potential clinical applications. α-bungarotoxin has been used as a molecular probe to study the structure and function of nAChRs (Nicotinic Acetylcholine Receptors). Acid-sensing ion channel (ASIC) isoforms 1a and 1b are inhibited by Mambalgins, derived from Black mamba venom, which hinders their function and provide an analgesic effect. α- Cobra toxin is a neurotoxin purified from Chinese cobra (Naja atra) binds to nAChRat the neuronal junction and causes an analgesic effect for moderate to severe pain. Some of the plants and their compounds have been shown to inhibit the activity of 3FTX, and their mechanisms of action are discussed.

Synthesis, Activity, and Application of Fluorescent Analogs of [D1G, Δ14Q]LvIC Targeting α6β4 Nicotinic Acetylcholine Receptor.

α6β4* nicotinic acetylcholine receptor (nAChR) (* represents the possible presence of additional subunits) is mainly distributed in the central and peripheral nervous system and is associated with neurological diseases, such as neuropathic pain; however, the ability to explore its function and distribution is limited due to the lack of pharmacological tools. As one of the analogs of α-conotoxin (α-CTx) LvIC from Conus lividus, [D1G, Δ14Q]LvIC (Lv) selectively and potently blocks α6/α3β4 nAChR (α6/α3 represents a chimera). Here, we synthesized three fluorescent analogs of Lv by connecting fluorescent molecules 6-carboxytetramethylrhodamine succinimidyl ester (6-TAMRA-SE, R), Cy3 NHS ester (Cy3, C) and BODIPY-FL NHS ester (BDP, B) to the N-terminus of the peptide and obtained Lv-R, Lv-C, and Lv-B, respectively. The potency and selectivity of three fluorescent peptides were evaluated using two-electrode voltage-clamp recording on nAChR subtypes expressed in Xenopus laevis oocytes, and the potency and selectivity of Lv-B were almost maintained with the half-maximal inhibition (IC50) of 64 nM. Then, we explored the stability of Lv-B in artificial cerebrospinal fluid and stained rat brain slices with Lv-B. The results indicated that the stability of Lv-B was slightly improved compared to that of native Lv. Additionally, we detected the distribution of the α6β4* nAChR subtype in the cerebral cortex using green fluorescently labeled peptide and fluorescence microscopy. Our findings not only provide a visualized pharmacological tool for exploring the distribution of the α6β4* nAChR subtype in various situ tissues and organs but also extend the application of α-CTx [D1G, Δ14Q]LvIC to demonstrate the involvement of α6β4 nAChR function in pathophysiology and pharmacology.

Genetic deletion of α7 nAChRs reduces hippocampal granule and pyramidal cell number in both sexes but impairs pattern separation in males only.

Neurogenesis within the dentate gyrus is thought to play an important role in cognitive processes such as reversal learning and pattern separation. The α7 nicotinic acetylcholine receptor (α7 nAChR) is expressed early in newly formed granule cells of the dentate gyrus, though its role in neurogenesis and related cognitive function is not fully understood. To better characterize relevant function of α7 nAChRs, we performed unbiased stereology to quantify hippocampal granule cells, pyramidal cells, and total volume and used a touchscreen operant spatial discrimination/reversal task to test pattern separation in a global α7 nAChR knockout mouse line. The knockout resulted in an ≈22% reduction in granule cells and a ≈ 20% reduction in pyramidal cells in both sexes, with no change in total hippocampal volume. However, the knockout impaired performance in the touchscreen task for males only. The sex-dependent difference in behavioral, but not stereological, results suggest a divergence in the structure-function relationship in males versus females. Detailed analyses revealed males were more biased by the initial reversal contingency relative to females indicating a potential source of the sex-specific interaction with the loss of α7 nAChRs. These findings argue that the α7 nAChR plays a critical role in hippocampal development, not just granule cell neurogenesis, and plays a sex-dependent role in cognitive function.

Ligand-Dependent Intramolecular Motion of Native Nicotinic Acetylcholine Receptors Determined in Living Myotube Cells via Diffracted X-ray Tracking.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that play an important role in signal transduction at the neuromuscular junction (NMJ). Movement of the nAChR extracellular domain following agonist binding induces conformational changes in the extracellular domain, which in turn affects the transmembrane domain and opens the ion channel. It is known that the surrounding environment, such as the presence of specific lipids and proteins, affects nAChR function. Diffracted X-ray tracking (DXT) facilitates measurement of the intermolecular motions of receptors on the cell membranes of living cells, including all the components involved in receptor function. In this study, the intramolecular motion of the extracellular domain of native nAChR proteins in living myotube cells was analyzed using DXT for the first time. We revealed that the motion of the extracellular domain in the presence of an agonist (e.g., carbamylcholine, CCh) was restricted by an antagonist (i.e., alpha-bungarotoxin, BGT).

Desensitizing nicotinic agents normalize tinnitus-related inhibitory dysfunction in the auditory cortex and ameliorate behavioral evidence of tinnitus.

Tinnitus impacts between 10-20% of the population. Individuals most troubled by their tinnitus have their attention bound to and are distracted by, their tinnitus percept. While numerous treatments to ameliorate tinnitus have been tried, no therapeutic approach has been clinically accepted. The present study used an established condition-suppression noise-exposure rat model of tinnitus to: (1) examine tinnitus-related changes in nAChR function of layer 5 pyramidal (PNs) and of vasoactive intestinal peptide (VIP) neurons in primary auditory cortex (A1) and (2) examine how the partial desensitizing nAChR agonists, sazetidine-A and varenicline, can act as potential therapeutic agents in the treatment of tinnitus. We posited that tinnitus-related changes in layer 5 nAChR responses may underpin the decline in attentional resources previously observed in this animal model (Brozoski et al., 2019). In vitro whole-cell patch-clamp studies previously revealed a significant tinnitus-related loss in nAChR-evoked excitatory postsynaptic currents from A1 layer 5 PNs. In contrast, VIP neurons from animals with behavioral evidence of tinnitus showed significantly increased nAChR-evoked excitability. Here we hypothesize that sazetidine-A and varenicline have therapeutic benefits for subjects who cannot divert their attention away from the phantom sound in their heads. We found that sazetidine-A or varenicline normalized tinnitus-related reductions in GABAergic input currents onto A1 layer 5 PNs. We then tested sazetidine-A and varenicline for the management of tinnitus using our tinnitus animal model. Subcutaneous injection of sazetidine-A or varenicline, 1 h prior to tinnitus testing, significantly decreased the rat's behavioral evidence of tinnitus in a dose-dependent manner. Collectively, these results support the need for additional clinical investigations of partial desensitizing nAChR agonists sazetidine-A and varenicline for the treatment of tinnitus.

Nicotine-mediated signaling in glomerular podocytes

Introduction: Although nicotine’s harmful effects on renal function are established, the precise cellular mechanisms of smoking-related damage are understudied. Smoking-Related Glomerulopathy (SRG) is a renal disease phenotype associated with smoking. This condition histologically mimics diabetic nephropathy, however, SRG patients present with proteinuria and renal insufficiency without diabetes. Here we investigated the acute and chronic nicotine-related oxidative and nitrosative stress in glomerular podocytes. We hypothesized that nicotine may promote nitrosative stress in the kidney cells through the rapid production of peroxynitrite (ONOO - ) and a decline in nitric oxide (NO) bioavailability. Methods: To test the hypothesis, we used a conditionally immortalized human podocyte line and confocal imaging to detect the production of ONOO - (Hydroxyl Radical and Peroxynitrite Sensor; HPF), intracellular Ca 2+ (Fluo-8), and NO (DAF-FM). The presence of nicotinic acetylcholine receptors (nAChR) receptor subunits in podocytes was confirmed with immunocytochemistry and live imaging using commercially available pharmacology. NOS activity was analyzed in response to Ang II with the specific blockers for NOS1 (NΩ-Propyl-L-arginine hydrochloride) and NOS2 (L-NIL) subunits. One-way ANOVA (OriginPro) was used for statistical analysis. Results: Immunostaining indicated that human and rat kidneys express nicotinic acetylcholine receptors (nAChR). Notably, we detected specific expression of α7 nAChR in glomerular podocytes. In podocytes, acute nAChR activation promoted the mobilization of intracellular Ca 2+ controlled by intracellular store activation, and fast ONOO - transients. Multiple consecutive applications of nicotine resulted in repeated intracellular Ca 2+ and ONOO - transients. Nicotine-mediated ONOO - response was efficiently blocked in the presence of superoxide dismutase (SOD). The application of specific α7 or α4β2, α2β4, α4β4 and α3β4 nAChR agonists elicited Ca 2+ transients but did not reproduce the ONOO - response to nicotine, suggesting that nitrosative processes may occur independently from Ca 2+ influx or nAChR function. Chronic exposure to nicotine (12-hrs) resulted in a significant decline in podocytes’ NO bioavailability (p<0.05). While under normal conditions, NO is produced primarily by NOS1 (70%), and the rest is attributed to NOS2, chronic nicotine exposure led to an elevation of NOS2 (75%) and a decline in NOS1 activity (p<0.001). We observed similar NOS remodeling under hyperglycemic conditions, suggesting similar nitrosative processes in response to nicotine and high glucose. Conclusions: Nicotine promotes superoxide-stimulated nitrosative stress and peroxynitrite formation in podocytes leading to decline in NO bioavailability and pathological activation of NOS2. R01 NIDDK DK126720 (OP), DK129227 (OP), HL148114 (DVI), NIH/NCATS/SCTR UL1TR001450/SCTR 2214 (OP). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.