Unraveling the molecular interactions between α7 nicotinic receptor and a RIC3 variant associated with backward speech.

Recent work putatively linked a rare genetic variant of the chaperone Resistant to Inhibitors of acetylcholinesterase (RIC3) (NM_024557.4:c.262G > A, NP_078833.3:p.G88R) to a unique ability to speak backwards, a language skill that is associated with exceptional working memory capacity. RIC3 is important for the folding, maturation, and functional expression of α7 nicotinic acetylcholine receptors (nAChR). We compared and contrasted the effects of RIC3G88R on assembly, cell surface expression, and function of human α7 receptors using fluorescent protein tagged α7 nAChR and Förster resonance energy transfer (FRET) microscopy imaging in combination with functional assays and 125I-α-bungarotoxin binding. As expected, the wild-type RIC3 protein was found to increase both cell surface and functional expression of α7 receptors. In contrast, the variant form of RIC3 decreased both. FRET analysis showed that RICG88R increased the interactions between RIC3 and α7 protein in the endoplasmic reticulum. These results provide interesting and novel data to show that a RIC3 variant alters the interaction of RIC3 and α7, which translates to decreased cell surface and functional expression of α7 nAChR.

Cholinergic Capsules and Academic Admonitions.

Herein, I intend to capture highlights shared with my academic and research colleagues over the 60 years I devoted initially to my graduate and postdoctoral training and then to academic endeavors starting as an assistant professor in a new medical school at the University of California, San Diego (UCSD). During this period, the Department of Pharmacology emerged from a division within the Department of Medicine to become the first basic science department, solely within the School of Medicine at UCSD in 1979. As part of the school's plans to reorganize and to retain me at UCSD, I was appointed as founding chair. Some years later in 2002, faculty, led largely within the Department of Pharmacology and by practicing pharmacists within UCSD Healthcare, started the independent Skaggs School of Pharmacy and Pharmaceutical Sciences with a doctor of pharmacy (PharmD) program, where I served as the founding dean. My career pathway, from working at my family-owned pharmacy to chairing a department in a school of medicine and then becoming the dean of a school of pharmacy at a research-intensive, student-centered institution, involved some risky decisions. But the academic, curricular, and accreditation challenges posed were met by a cadre of creative faculty colleagues. I offer my experiences to individuals confronted with a multiplicity of real or imagined opportunities in academic health sciences, the related pharmaceutical industry, and government oversight agencies.

The angiogenic role of the alpha 9-nicotinic acetylcholine receptor in triple-negative breast cancers.

Nicotine acts as an angiogenic factor by stimulating endogenous cholinergic pathways. Several subtypes of nicotinic acetylcholine receptors (nAChRs) have been demonstrated to be closely correlated to the formation and progression of different types of cancers. Recently, several studies have found that nicotinic acetylcholine receptors α9 (α9-nAChRs) are highly expressed in breast tumors, especially in tumors derived from patients diagnosed at advanced stages. In vitro studies have demonstrated that activation of α9-nAChRs is associated with increased proliferation and migration of breast cancer. To study the tumor-promoting role of α9-nAChRs in breast cancers, we generated a novel anti-α9-nAChR and methoxy-polyethylene glycol (mPEG) bispecific antibody (α9 BsAb) for dissecting the molecular mechanism on α9-nAChR-mediated tumor progression. Unexpectedly, we discovered the angiogenic role of α9-nAChR in nicotine-induced neovascularization of tumors. It revealed α9 BsAbs reduced nicotine-induced endothelial cell tube formation, blood vessel development in Matrigel plug assay and angiogenesis in microtube array membrane murine model (MTAMs). To unbraid the molecular mechanism of α9-nAChR in nicotine-mediated angiogenesis, the α9 BsAbs were applied and revealed the inhibitory roles in nicotine-induced production of hypoxia-inducible factor-2 alpha (HIF-2α), vascular endothelial growth factor A (VEGF-A), phosphorylated vascular endothelial growth factor receptor 2 (p-VEGFR2), vascular endothelial growth factor receptor 2 (VEGFR2) and matrix metalloproteinase-9 (MMP9) from triple-negative breast cancer cells (MDA-MB-231), suggesting α9-nAChRs played an important role in nicotine-induced angiogenesis. To confirm our results, the shRNA targeting α9-nAChRs was designed and used to silence α9-nAChR expression and then evaluated the angiogenic role of α9-nAChRs. The results showed α9 shRNA also played an inhibitory effect in blocking the nicotine-induced angiogenic signaling. Taken together, α9-nAChR played a critical role in nicotine-induced angiogenesis and this bispecific antibody (α9 BsAb) may serve as a potential therapeutic candidate for treatments of the α9 positive cancers.

Expression and function of nicotinic acetylcholine receptors in specific neuronal populations: Focus on striatal and prefrontal circuits.

Nicotinic acetylcholine receptors (nAChRs) are widely expressed in the central nervous system and play an important role in the control of neural functions including neuronal activity, transmitter release and synaptic plasticity. Although the common subtypes of nAChRs are abundantly expressed throughout the brain, their expression in different brain regions and by individual neuronal types is not homogeneous or incidental. In recent years, several studies have emerged showing that particular subtypes of nAChRs are expressed by specific neuronal populations in which they have major influence on the activity of local circuits and behavior. It has been demonstrated that even nAChRs expressed by relatively rare neuronal types can induce significant changes in behavior and contribute to pathological processes. Depending on the identity and connectivity of the particular nAChRs-expressing neuronal populations, the activation of nAChRs can have distinct or even opposing effects on local neuronal signaling. In this review, we will summarize the available literature describing the expression of individual nicotinic subunits by different neuronal types in two crucial brain regions, the striatum and the prefrontal cortex. The review will also briefly discuss nicotinic expression in non-neuronal, glial cells, as they cannot be ignored as potential targets of nAChRs-modulating drugs. The final section will discuss options that could allow us to target nAChRs in a neuronal-type-specific manner, not only in the experimental field, but also eventually in clinical practice.

Auxiliary protein and chaperone regulation of neuronal nicotinic receptor subtype expression and function.

Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of pentameric, ligand-gated ion channels that are located on the surface of neurons and non-neuronal cells and have multiple physiological and pathophysiological functions. In order to reach the cell surface, many nAChR subtypes require the help of chaperone and/or auxiliary/accessory proteins for their assembly, trafficking, pharmacological modulation, and normal functioning in vivo. The use of powerful genome-wide cDNA screening has led to the identification and characterisation of the molecules and mechanisms that participate in the assembly and trafficking of receptor subtypes, including chaperone and auxiliary or accessory proteins. The aim of this review is to describe the latest findings concerning nAChR chaperones and auxiliary proteins and pharmacological chaperones, and how some of them control receptor biogenesis or regulate channel activation and pharmacology. Some auxiliary proteins are subtype selective, some regulate various subtypes, and some not only modulate nAChRs but also target other receptors and signalling pathways. We also discuss how changes in auxiliary proteins may be involved in nAChR dysfunctions.

Swift regulation of nicotinic acetylcholine receptors (nAChRs) and glutathione S-transferase (GST) enables the rapid detoxification of thiacloprid in pine sawyer beetles.

Thiacloprid, a neonicotinoid insecticide, has become one of the major control agents for the pine sawyer beetle, Monochamus alternatus Hope, however, the mechanism of detoxification is unknown. We demonstrate that glutathione S-transferases (GSTs) and nicotinic acetylcholine receptors (nAChRs) are involved in the rapid detoxification of thiacloprid in M. alternatus larvae. The activity of detoxification enzyme GSTs was significantly higher, while the activity of acetylcholinesterase (AChE) was inhibited under thiacloprid exposure. The inhibition of AChE activity led to lethal over-stimulation of the cholinergic synapse, which was then released by the rapid downregulation of nAChRs. Meanwhile, GSTs were overexpressed to detoxify thiacloprid accordingly. A total of 3 nAChR and 12 GST genes were identified from M. alternatus, among which ManAChRα2 and MaGSTs1 were predicted to confer thiacloprid tolerance. RNA interference (RNAi) was subsequently conducted to confirm the function of ManAChRα2 and MaGSTs1 genes in thiacloprid detoxification. The successful knock-down of the ManAChRα2 gene led to lower mortality of M. alternatus under LC30 thiacloprid treatment, and the suppression of the MaGSTs1 gene increased the mortality rate of M. alternatus. However, the mortality rate has no significant difference with controls when thiacloprid was fed together with both dsMaGSTs1 and dsManAChRα2. Molecular docking modeled the molecular basis for interaction between MaGSTs1/ManAChR and thiacloprid. This study highlights the important roles that ManAChRα2 and MaGSTs1 genes play in thiacloprid detoxification through transcriptional regulation and enzymatic metabolization, and proposes a new avenue for integrated pest management that combines pesticides and RNAi technology as an efficient strategy for M. alternatus control.

Assessing potentiation of the (α4)3(ß2)2 nicotinic acetylcholine receptor by the allosteric agonist CMPI.

The extracellular domain of the nicotinic acetylcholine receptor isoforms formed by three α4 and two ß2 subunits ((α4)3(ß2)2 nAChR) harbors two high-affinity "canonical" acetylcholine (ACh)-binding sites located in the two α4:ß2 intersubunit interfaces and a low-affinity "noncanonical" ACh-binding site located in the α4:α4 intersubunit interface. In this study, we used ACh, cytisine, and nicotine (which bind at both the α4:α4 and α4:ß2 interfaces), TC-2559 (which binds at the α4:ß2 but not at the α4:α4 interface), and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI, which binds at the α4:α4 but not at the α4:ß2 interface), to investigate the binding and gating properties of CMPI at the α4:α4 interface. We recorded whole-cell currents from Xenopus laevis oocytes expressing (α4)3(ß2)2 nAChR in response to applications of these ligands, alone or in combination. The electrophysiological data were analyzed in the framework of a modified Monod-Wyman-Changeux allosteric activation model. We show that CMPI is a high-affinity, high-efficacy agonist at the α4:α4 binding site and that its weak direct activating effect is accounted for by its inability to productively interact with the α4:ß2 sites. The data presented here enhance our understanding of the functional contributions of ligand binding at the α4:α4 subunit interface to (α4)3(ß2)2 nAChR-channel gating. These findings support the potential use of α4:α4 specific ligands to increase the efficacy of the neurotransmitter ACh in conditions associated with decline in nAChRs activity in the brain.

Deletion induced splicing in RIC3 drives nicotinic acetylcholine receptor regulation with implications for endoplasmic reticulum stress in human astrocytes.

Nicotinic acetylcholine receptor (nAChR) dysregulation in astrocytes is reported in neurodegenerative disorders. Modulation of nAChRs through agonists confers protection to astrocytes from stress but regulation of chaperones involved in proteostasis with pathological implications is unclear. Resistance to inhibitors of cholinesterase 3 (RIC3), a potential chaperone of nAChRs is poorly studied in humans. We characterized RIC3 in astrocytes derived from an isogenic wild-type and Cas9 edited "del" human iPSC line harboring a 25 bp homozygous deletion in exon2. Altered RIC3 transcript ratio due to deletion induced splicing and an unexpected gain of α7nAChR expression were observed in "del" astrocytes. Transcriptome analysis showed higher expression of neurotransmitter/G-protein coupled receptors mediated by cAMP and calcium/calmodulin-dependent kinase signaling with increased cytokines/glutamate secretion. Functional implications examined using tunicamycin induced ER stress in wild-type astrocyte stress model showed cell cycle arrest, RIC3 upregulation, reduction in α7nAChR membrane levels but increased α4nAChR membrane expression. Conversely, tunicamycin-treated "del" astrocytes showed a comparatively higher α4nAChR membrane expression and upsurged cAMP signaling. Furthermore, reduced expression of stress markers CHOP, phospho-PERK and lowered XBP1 splicing in western blot and qPCR, validated by proteome-based pathway analysis indicated lowered disease severity. Findings indicate (i) a complex RNA regulatory mechanism via exonic deletion induced splicing; (ii) RIC-3 as a disordered protein having contrasting effects on co-expressed nAChR subtypes under basal/stress conditions; and (iii) RIC3 as a potential drug target against ER stress in astrocytes for neurodegenerative/nicotine-related brain disorders. Cellular rescue mechanism through deletion induced exon skipping may encourage ASO-based therapies for tauopathies.

Neonicotinoid Insecticides: Molecular Targets, Resistance, and Toxicity.

Neonicotinoids have been used to protect crops and animals from insect pests since the 1990s, but there are concerns regarding their adverse effects on nontarget organisms, notably on bees. Enhanced resistance to neonicotinoids in pests is becoming well documented. We address the current understanding of neonicotinoid target site interactions, selectivity, and metabolism not only in pests but also in beneficial insects such as bees. The findings are relevant to the management of both neonicotinoids and the new generation of pesticides targeting insect nicotinic acetylcholine receptors.

Induction of tetraspanin 13 contributes to the synergistic anti-inflammatory effects of parasympathetic and sympathetic stimulation in macrophages.

The autonomic nervous system plays an important role in the regulation of peripheral inflammation. Sympathetic nervous activation stimulates inflammatory gene expression and cytokines, whereas parasympathetic nervous activation suppresses the production of inflammatory cytokines by immune cells. However, most studies on the relationship between the autonomic nervous system and immune processes have analyzed a single branch of the autonomic nerves in isolation. Therefore, this study aimed to examine the effects of sympathetic and parasympathetic stimulation on macrophages, which are controlled by autonomic regulation. Macrophages were stimulated with lipopolysaccharide (LPS) to induce TNF-α. Then, the effects of ß2 adrenergic receptor and α7 nicotinic acetylcholine receptor activation on TNF-α production were assessed using concentration-dependent assays. RNA-seq data were also used to identify genes whose expression was enhanced by parasympathetic and sympathetic stimulation. The simultaneous activation of ß2 adrenergic receptors and α7 nicotinic acetylcholine receptors suppressed LPS-induced TNF-α production in a concentration-dependent manner. Moreover, simultaneous activation of these receptors had synergistic anti-inflammatory effects and induced Tspan13 expression, thereby contributing to anti-inflammatory mechanisms in macrophages. Our study revealed the synergistic anti-inflammatory effects of the parasympathetic and sympathetic stimulation of macrophages. Our results suggest that targeting both sympathetic and parasympathetic signaling is a promising therapeutic approach for inflammatory diseases.

Expression of subunits of an insecticide target receptor varies across tissues, life stages, castes, and species of social bees.

Global losses of insects jeopardize ecosystem stability and crop pollination. Robust evidence indicates that insecticides have contributed to these losses. Notably, insecticides targeting nicotinic acetylcholine receptors (nAChRs) have neurotoxic effects on beneficial insects. Because each nAChR consists of five subunits, the alternative arrangements of subunits could create a multitude of receptors differing in structure and function. Therefore, understanding whether the use of subunits varies is essential for evaluating and predicting the effects of insecticides targeting such receptors. To better understand how the use and composition of nAChRs differ within and between insect pollinators, we analysed RNA-seq gene expression data from tissues and castes of Apis mellifera honey bees and life stages and castes of the Bombus terrestris bumble bees. We reveal that all analysed tissues express nAChRs and that relative expression levels of nAChR subunits vary widely across almost all comparisons. Our work thus shows fine-tuned spatial and temporal expression of nAChRs. Given that coexpression of subunits underpins the compositional diversity of functional receptors and that the affinities of insecticides depend on nAChR composition, our findings provide a likely mechanism for the various damaging effects of nAChR-targeting insecticides on insects. Furthermore, our results indicate that the appraisal of insecticide risks should carefully consider variation in molecular targets.

The role of nicotinic receptors in alcohol consumption.

The use of alcohol causes significant morbidity and mortality across the globe. Alcohol use disorder (AUD) is defined by the excessive use of this drug despite a negative impact on the individual's life. While there are currently medications available to treat AUD, they have limited efficacy and several side effects. As such, it is essential to continue to look for novel therapeutics. One target for novel therapeutics is nicotinic acetylcholine receptors (nAChRs). Here we systematically review the literature on the involvement of nAChRs in alcohol consumption. Data from both genetic and pharmacology studies provide evidence that nAChRs modulate alcohol intake. Interestingly, pharmacological modulation of all nAChR subtypes examined can decrease alcohol consumption. The reviewed literature demonstrates that nAChRs should continue to be investigated as novel therapeutics for AUD.

Pathophysiology of nAChRs: Limbic circuits and related disorders.

Human epidemiological studies have identified links between nicotine intake and stress disorders, including anxiety, depression and PTSD. Here we review the clinical evidence for activation and desensitization of nicotinic acetylcholine receptors (nAChRs) relevant to affective disorders. We go on to describe clinical and preclinical pharmacological studies suggesting that nAChR function may be involved in the etiology of anxiety and depressive disorders, may be relevant targets for medication development, and may contribute to the antidepressant efficacy of non-nicotinic therapeutics. We then review what is known about nAChR function in a subset of limbic system areas (amygdala, hippocampus and prefrontal cortex), and how this contributes to stress-relevant behaviors in preclinical models that may be relevant to human affective disorders. Taken together, the preclinical and clinical literature point to a clear role for ACh signaling through nAChRs in regulation of behavioral responses to stress. Disruption of nAChR homeostasis is likely to contribute to the psychopathology observed in anxiety and depressive disorders. Targeting specific nAChRs may therefore be a strategy for medication development to treat these disorders or to augment the efficacy of current therapeutics.

Role of neuronal nicotinic acetylcholine receptors in cannabinoid dependence.

Cannabis is among the most widely consumed psychoactive drugs around the world and cannabis use disorder (CUD) has no current approved pharmacological treatment. Nicotine and cannabis are commonly co-used which suggests there to be overlapping neurobiological actions supported primarily by the co-distribution of both receptor systems in the brain. There appears to be strong rationale to explore the role that nicotinic receptors play in cannabinoid dependence. Preclinical studies suggest that the ɑ7 nAChR subtype may play a role in modulating the reinforcing and discriminative stimulus effects of cannabinoids, while the ɑ4ß2 * nAChR subtype may be involved in modulating the motor and sedative effects of cannabinoids. Preclinical and human genetic studies point towards a potential role of the ɑ5, ɑ3, and ß4 nAChR subunits in CUD, while human GWAS studies strongly implicate the ɑ2 subunit as playing a role in CUD susceptibility. Clinical studies suggest that current smoking cessation agents, such as varenicline and bupropion, may also be beneficial in treating CUD, although more controlled studies are necessary. Additional behavioral, molecular, and mechanistic studies investigating the role of nAChR in the modulation of the pharmacological effects of cannabinoids are needed.

Signaling of nicotinic acetylcholine receptors in mononuclear phagocytes.

Nicotinic acetylcholine receptors are not only expressed by the nervous system and at the neuro-muscular junction but also by mononuclear phagocytes, which belong to the innate immune system. Mononuclear phagocyte is an umbrella term for monocytes, macrophages, and dendritic cells. These cells play pivotal roles in host defense against infection but also in numerous often debilitating diseases that are characterized by exuberant inflammation. Nicotinic acetylcholine receptors of the neuronal type dominate in these cells, and their stimulation is mainly associated with anti-inflammatory effects. Although the cholinergic modulation of mononuclear phagocytes is of eminent clinical relevance for the prevention and treatment of inflammatory diseases and neuropathic pain, we are only beginning to understand the underlying mechanisms on the molecular level. The purpose of this review is to report and critically discuss the current knowledge on signal transduction mechanisms elicited by nicotinic acetylcholine receptors in mononuclear phagocytes.

Central and peripheral actions of nicotine that influence blood glucose homeostasis and the development of diabetes.

Cigarette smoking has long been recognized as a risk factor for type 2 diabetes (T2D), although the precise causal mechanisms underlying this relationship remain poorly understood. Recent evidence suggests that nicotine, the primary reinforcing component in tobacco, may play a pivotal role in connecting cigarette smoking and T2D. Extensive research conducted in both humans and animals has demonstrated that nicotine can elevate blood glucose levels, disrupt glucose homeostasis, and induce insulin resistance. The review aims to elucidate the genetic variants of nicotinic acetylcholine receptors associated with diabetes risk and provide a comprehensive overview of the available data on the mechanisms through which nicotine influences blood glucose homeostasis and the development of diabetes. Here we emphasize the central and peripheral actions of nicotine on the release of glucoregulatory hormones, as well as its effects on glucose tolerance and insulin sensitivity. Notably, the central actions of nicotine within the brain, which encompass both insulin-dependent and independent mechanisms, are highlighted as potential targets for intervention strategies in diabetes management.

Advances in small molecule selective ligands for heteromeric nicotinic acetylcholine receptors.

The study of nicotinic acetylcholine receptors (nAChRs) has significantly progressed in the last decade, due to a) the improved techniques available for structural studies; b) the identification of ligands interacting at orthosteric and allosteric recognition sites on the nAChR proteins, able to tune channel conformational states; c) the better functional characterization of receptor subtypes/subunits and their therapeutic potential; d) the availability of novel pharmacological agents able to activate or block nicotinic-mediated cholinergic responses with subtype or stoichiometry selectivity. The copious literature on nAChRs is related to the pharmacological profile of new, promising subtype selective derivatives as well as the encouraging preclinical and early clinical evaluation of known ligands. However, recently approved therapeutic derivatives are still missing, and examples of ligands discontinued in advanced CNS clinical trials include drug candidates acting at both neuronal homomeric and heteromeric receptors. In this review, we have selected heteromeric nAChRs as the target and comment on literature reports of the past five years dealing with the discovery of new small molecule ligands or the advanced pharmacological/preclinical investigation of more promising compounds. The results obtained with bifunctional nicotinic ligands and a light-activated ligand as well as the applications of promising radiopharmaceuticals for heteromeric subtypes are also discussed.

Characterisation of Elevenin-Vc1 from the Venom of Conus victoriae: A Structural Analogue of α-Conotoxins.

Elevenins are peptides found in a range of organisms, including arthropods, annelids, nematodes, and molluscs. They consist of 17 to 19 amino acid residues with a single conserved disulfide bond. The subject of this study, elevenin-Vc1, was first identified in the venom of the cone snail Conus victoriae (Gen. Comp. Endocrinol. 2017, 244, 11-18). Although numerous elevenin sequences have been reported, their physiological function is unclear, and no structural information is available. Upon intracranial injection in mice, elevenin-Vc1 induced hyperactivity at doses of 5 or 10 nmol. The structure of elevenin-Vc1, determined using nuclear magnetic resonance spectroscopy, consists of a short helix and a bend region stabilised by the single disulfide bond. The elevenin-Vc1 structural fold is similar to that of α-conotoxins such as α-RgIA and α-ImI, which are also found in the venoms of cone snails and are antagonists at specific subtypes of nicotinic acetylcholine receptors (nAChRs). In an attempt to mimic the functional motif, Asp-Pro-Arg, of α-RgIA and α-ImI, we synthesised an analogue, designated elevenin-Vc1-DPR. However, neither elevenin-Vc1 nor the analogue was active at six different human nAChR subtypes (α1ß1εδ, α3ß2, α3ß4, α4ß2, α7, and α9α10) at 1 µM concentrations.

Substitution of D-Arginine at Position 11 of α-RgIA Potently Inhibits α7 Nicotinic Acetylcholine Receptor.

Conotoxins are a class of disulfide-rich peptides found in the venom of cone snails, which have attracted considerable attention in recent years due to their potent activity on ion channels and potential for therapeutics. Among them, α-conotoxin RgIA, a 13-residue peptide, has shown great promise as a potent inhibitor of α9α10 nAChRs for pain management. In this study, we investigated the effect of substituting the naturally occurring L-type arginine at position 11 of the RgIA sequence with its D-type amino acid. Our results indicate that this substitution abrogated the ability of RgIA to block α9α10 nAChRs, but instead endowed the peptide with the ability to block α7 nAChR activity. Structural analyses revealed that this substitution induced significant alteration of the secondary structure of RgIA[11r], which consequently affected its activity. Our findings underscore the potential of D-type amino acid substitution as a promising strategy for designing novel conotoxin-based ligands targeting different types of nAChRs.

Synthesis and Biological Activity of Novel α-Conotoxins Derived from Endemic Polynesian Cone Snails.

α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii. Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3; 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 ß2ß3 subtype (IC50 = 177 nM). Overall, this study contributes to a better understanding of the structure-activity relationships of α-conotoxins, which may help in the design of more selective tools.