Spermatogonial stem cells in the 129 inbred strain exhibit unique requirements for self-renewal.

Spermatogonial stem cells (SSCs) undergo self-renewal division to sustain spermatogenesis. Although it is possible to derive SSC cultures in most mouse strains, SSCs from a 129 background never proliferate under the same culture conditions, suggesting they have distinct self-renewal requirements. Here, we established long-term culture conditions for SSCs from mice of the 129 background (129 mice). An analysis of 129 testes showed significant reduction of GDNF and CXCL12, whereas FGF2, INHBA and INHBB were higher than in testes of C57BL/6 mice. An analysis of undifferentiated spermatogonia in 129 mice showed higher expression of Chrna4, which encodes an acetylcholine (Ach) receptor component. By supplementing medium with INHBA and Ach, SSC cultures were derived from 129 mice. Following lentivirus transduction for marking donor cells, transplanted cells re-initiated spermatogenesis in infertile mouse testes and produced transgenic offspring. These results suggest that the requirements of SSC self-renewal in mice are diverse, which has important implications for understanding self-renewal mechanisms in various animal species.

Wnt signaling regulates acetylcholine receptor translocation and synaptic plasticity in the adult nervous system.

The adult nervous system is plastic, allowing us to learn, remember, and forget. Experience-dependent plasticity occurs at synapses--the specialized points of contact between neurons where signaling occurs. However, the mechanisms that regulate the strength of synaptic signaling are not well understood. Here, we define a Wnt-signaling pathway that modifies synaptic strength in the adult nervous system by regulating the translocation of one class of acetylcholine receptors (AChRs) to synapses. In Caenorhabditis elegans, we show that mutations in CWN-2 (Wnt ligand), LIN-17 (Frizzled), CAM-1 (Ror receptor tyrosine kinase), or the downstream effector DSH-1 (disheveled) result in similar subsynaptic accumulations of ACR-16/α7 AChRs, a consequent reduction in synaptic current, and predictable behavioral defects. Photoconversion experiments revealed defective translocation of ACR-16/α7 to synapses in Wnt-signaling mutants. Using optogenetic nerve stimulation, we demonstrate activity-dependent synaptic plasticity and its dependence on ACR-16/α7 translocation mediated by Wnt signaling via LIN-17/CAM-1 heteromeric receptors.

Ion transport in muscle acetylcholine receptor maintained by conserved salt bridges between the pore and lipid membrane.

Pores through ion channels rapidly transport small inorganic ions along their electrochemical gradients. Here, applying single-channel electrophysiology and mutagenesis to the archetypal muscle nicotinic acetylcholine receptor (AChR) channel, we show that a conserved pore-peripheral salt bridge partners with those in the other subunits to regulate ion transport. Disrupting the salt bridges in all five receptor subunits greatly decreases the amplitude of the unitary current and increases its fluctuations. However, disrupting individual salt bridges has unequal effects that depend on the structural status of the other salt bridges. The AChR ε- and δ-subunits are structurally unique in harboring a putative palmitoylation site near each salt bridge and bordering the lipid membrane. The effects of disrupting the palmitoylation sites mirror those of disrupting the salt bridges, but the effect of disrupting either of these structures depends on the structural status of the other. Thus, rapid ion transport through the AChR channel is maintained by functionally interdependent salt bridges linking the pore to the lipid membrane.

Dual mutations in the whitefly nicotinic acetylcholine receptor ß1 subunit confer target-site resistance to multiple neonicotinoid insecticides.

Neonicotinoid insecticides, which target insect nicotinic acetylcholine receptors (nAChRs), have been widely and intensively used to control the whitefly, Bemisia tabaci, a highly damaging, globally distributed, crop pest. This has inevitably led to the emergence of populations with resistance to neonicotinoids. However, to date, there have been no reports of target-site resistance involving mutation of B. tabaci nAChR genes. Here we characterize the nAChR subunit gene family of B. tabaci and identify dual mutations (A58T&R79E) in one of these genes (BTß1) that confer resistance to multiple neonicotinoids. Transgenic D. melanogaster, where the native nAChR Dß1 was replaced with BTß1A58T&R79E, were significantly more resistant to neonicotinoids than flies where Dß1 were replaced with the wildtype BTß1 sequence, demonstrating the causal role of the mutations in resistance. The two mutations identified in this study replace two amino acids that are highly conserved in >200 insect species. Three-dimensional modelling suggests a molecular mechanism for this resistance, whereby A58T forms a hydrogen bond with the R79E side chain, which positions its negatively-charged carboxylate group to electrostatically repulse a neonicotinoid at the orthosteric site. Together these findings describe the first case of target-site resistance to neonicotinoids in B. tabaci and provide insight into the molecular determinants of neonicotinoid binding and selectivity.

Functional impact of subunit composition and compensation on Drosophila melanogaster nicotinic receptors-targets of neonicotinoids.

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. We recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. We show that the Dα3 subunit co-exists with Dα1, Dα2, Dß1, and Dß2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dß3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dß3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.

Pathogenic residue insertion in neuronal nicotinic receptor alters intra- and inter-subunit interactions that tune channel gating.

We describe molecular-level functional changes in the α4ß2 nicotinic acetylcholine receptor by a leucine residue insertion in the M2 transmembrane domain of the α4 subunit associated with sleep-related hyperkinetic epilepsy. Measurements of agonist-elicited single-channel currents reveal the primary effect is to stabilize the open channel state, while the secondary effect is to promote reopening of the channel. These dual effects prolong the durations of bursts of channel openings equally for the two major stoichiometric forms of the receptor, (α4)2(ß2)3 and (α4)3(ß2)2, indicating the functional impact is independent of mutant copy number per receptor. Altering the location of the residue insertion within M2 shows that functionally pivotal structures are confined to a half turn of the M2 α-helix. Residue substitutions within M2 and surrounding α-helices reveal that both intrasubunit and intersubunit interactions mediate the increase in burst duration. These interactions impacting burst duration depend linearly on the size and hydrophobicity of the substituting residue. Together, the results reveal a novel structural region of the α4ß2 nicotinic acetylcholine receptor in which interhelical interactions tune the stability of the open channel state.

Analogs of α-conotoxin PnIC selectively inhibit α7ß2- over α7-only subtype nicotinic acetylcholine receptors via a novel allosteric mechanism.

This study was undertaken to identify and characterize the first ligands capable of selectively identifying nicotinic acetylcholine receptors containing α7 and ß2 subunits (α7ß2-nAChR subtype). Basal forebrain cholinergic neurons express α7ß2-nAChR. Here, they appear to mediate neuronal dysfunction induced by the elevated levels of oligomeric amyloid-ß associated with early Alzheimer's disease. Additional work indicates that α7ß2-nAChR are expressed across several further critically important cholinergic and GABAergic neuronal circuits within the central nervous system. Further studies, however, are significantly hindered by the inability of currently available ligands to distinguish heteromeric α7ß2-nAChR from the closely related and more widespread homomeric α7-only-nAChR subtype. Functional screening using two-electrode voltage-clamp electrophysiology identified a family of α7ß2-nAChR-selective analogs of α-conotoxin PnIC (α-CtxPnIC). A combined electrophysiology, functional kinetics, site-directed mutagenesis, and molecular dynamics approach was used to further characterize the α7ß2-nAChR selectivity and site of action of these α-CtxPnIC analogs. We determined that α7ß2-nAChR selectivity of α-CtxPnIC analogs arises from interactions at a site distinct from the orthosteric agonist-binding site shared between α7ß2- and α7-only-nAChR. As numerous previously identified α-Ctx ligands are competitive antagonists of orthosteric agonist-binding sites, this study profoundly expands the scope of use of α-Ctx ligands (which have already provided important nAChR research and translational breakthroughs). More immediately, analogs of α-CtxPnIC promise to enable, for the first time, both comprehensive mapping of the distribution of α7ß2-nAChR and detailed investigations of their physiological roles.

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.

Nicotinic acetylcholine receptor modulator insecticides act on diverse receptor subtypes with distinct subunit compositions.

Insect nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels mainly expressed in the central nervous system of insects. They are the directed targets of many insecticides, including neonicotinoids, which are the most widely used insecticides in the world. However, the development of resistance in pests and the negative impacts on bee pollinators affect the application of insecticides and have created a demand for alternatives. Thus, it is very important to understand the mode of action of these insecticides, which is not fully understood at the molecular level. In this study, we systematically examined the susceptibility of ten Drosophila melanogaster nAChR subunit mutants to eleven insecticides acting on nAChRs. Our results showed that there are several subtypes of nAChRs with distinct subunit compositions that are responsible for the toxicity of different insecticides. At least three of them are the major molecular targets of seven structurally similar neonicotinoids in vivo. Moreover, spinosyns may act exclusively on the α6 homomeric pentamers but not any other nAChRs. Behavioral assays using thermogenetic tools further confirmed the bioassay results and supported the idea that receptor activation rather than inhibition leads to the insecticidal effects of neonicotinoids. The present findings reveal native nAChR subunit interactions with various insecticides and have important implications for the management of resistance and the development of novel insecticides targeting these important ion channels.

Identification of genetic risk loci and prioritization of genes and pathways for myasthenia gravis: a genome-wide association study.

Myasthenia gravis is a chronic autoimmune disease characterized by autoantibody-mediated interference of signal transmission across the neuromuscular junction. We performed a genome-wide association study (GWAS) involving 1,873 patients diagnosed with acetylcholine receptor antibody-positive myasthenia gravis and 36,370 healthy individuals to identify disease-associated genetic risk loci. Replication of the discovered loci was attempted in an independent cohort from the UK Biobank. We also performed a transcriptome-wide association study (TWAS) using expression data from skeletal muscle, whole blood, and tibial nerve to test the effects of disease-associated polymorphisms on gene expression. We discovered two signals in the genes encoding acetylcholine receptor subunits that are the most common antigenic target of the autoantibodies: a GWAS signal within the cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) gene and a TWAS association with the cholinergic receptor nicotinic beta 1 subunit (CHRNB1) gene in normal skeletal muscle. Two other loci were discovered on 10p14 and 11q21, and the previous association signals at PTPN22, HLA-DQA1/HLA-B, and TNFRSF11A were confirmed. Subgroup analyses demonstrate that early- and late-onset cases have different genetic risk factors. Genetic correlation analysis confirmed a genetic link between myasthenia gravis and other autoimmune diseases, such as hypothyroidism, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes. Finally, we applied Priority Index analysis to identify potentially druggable genes/proteins and pathways. This study provides insight into the genetic architecture underlying myasthenia gravis and demonstrates that genetic factors within the loci encoding acetylcholine receptor subunits contribute to its pathogenesis.

Neurobiological Mechanisms of Nicotine Reward and Aversion.

Neuronal nicotinic acetylcholine receptors (nAChRs) regulate the rewarding actions of nicotine contained in tobacco that establish and maintain the smoking habit. nAChRs also regulate the aversive properties of nicotine, sensitivity to which decreases tobacco use and protects against tobacco use disorder. These opposing behavioral actions of nicotine reflect nAChR expression in brain reward and aversion circuits. nAChRs containing α4 and ß2 subunits are responsible for the high-affinity nicotine binding sites in the brain and are densely expressed by reward-relevant neurons, most notably dopaminergic, GABAergic, and glutamatergic neurons in the ventral tegmental area. High-affinity nAChRs can incorporate additional subunits, including ß3, α6, or α5 subunits, with the resulting nAChR subtypes playing discrete and dissociable roles in the stimulatory actions of nicotine on brain dopamine transmission. nAChRs in brain dopamine circuits also participate in aversive reactions to nicotine and the negative affective state experienced during nicotine withdrawal. nAChRs containing α3 and ß4 subunits are responsible for the low-affinity nicotine binding sites in the brain and are enriched in brain sites involved in aversion, including the medial habenula, interpeduncular nucleus, and nucleus of the solitary tract, brain sites in which α5 nAChR subunits are also expressed. These aversion-related brain sites regulate nicotine avoidance behaviors, and genetic variation that modifies the function of nAChRs in these sites increases vulnerability to tobacco dependence and smoking-related diseases. Here, we review the molecular, cellular, and circuit-level mechanisms through which nicotine elicits reward and aversion and the adaptations in these processes that drive the development of nicotine dependence. SIGNIFICANCE STATEMENT: Tobacco use disorder in the form of habitual cigarette smoking or regular use of other tobacco-related products is a major cause of death and disease worldwide. This article reviews the actions of nicotine in the brain that contribute to tobacco use disorder.

SARS-CoV-2 spike ectodomain targets α7 nicotinic acetylcholine receptors.

Virus entry into animal cells is initiated by attachment to target macromolecules located on host cells. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) trimeric spike glycoprotein targets host angiotensin converting enzyme 2 to gain cellular access. The SARS-CoV-2 glycoprotein contains a neurotoxin-like region that has sequence similarities to the rabies virus and the HIV glycoproteins, as well as to snake neurotoxins, which interact with nicotinic acetylcholine receptor (nAChR) subtypes via this region. Using a peptide of the neurotoxin-like region of SARS-CoV-2 (SARS-CoV-2 glycoprotein peptide [SCoV2P]), we identified that this area moderately inhibits α3ß2, α3ß4, and α4ß2 subtypes, while potentiating and inhibiting α7 nAChRs. These nAChR subtypes are found in target tissues including the nose, lung, central nervous system, and immune cells. Importantly, SCoV2P potentiates and inhibits ACh-induced α7 nAChR responses by an allosteric mechanism, with nicotine enhancing these effects. Live-cell confocal microscopy was used to confirm that SCoV2P interacts with α7 nAChRs in transfected neuronal-like N2a and human embryonic kidney 293 cells. The SARS-CoV-2 ectodomain functionally potentiates and inhibits the α7 subtype with nanomolar potency. Our functional findings identify that the α7 nAChR is a target for the SARS-CoV-2 glycoprotein, providing a new aspect to our understanding of SARS-CoV-2 and host cell interactions, in addition to disease pathogenesis.

α7 nicotinic receptors play a role in regulation of cardiac hemodynamics.

The α7 nicotinic receptors (NR) have been confirmed in the heart but their role in cardiac functions has been contradictory. To address these contradictory findings, we analyzed cardiac functions in α7 NR knockout mice (α7-/-) in vivo and ex vivo in isolated hearts. A standard limb leads electrocardiogram was used, and the pressure curves were recorded in vivo, in Arteria carotis and in the left ventricle, or ex vivo, in the left ventricle of the spontaneously beating isolated hearts perfused following Langedorff's method. Experiments were performed under basic conditions, hypercholinergic conditions, and adrenergic stress. The relative expression levels of α and ß NR subunits, muscarinic receptors, ß1 adrenergic receptors, and acetylcholine life cycle markers were determined using RT-qPCR. Our results revealed a prolonged QT interval in α7-/- mice. All in vivo hemodynamic parameters were preserved under all studied conditions. The only difference in ex vivo heart rate between genotypes was the loss of bradycardia in prolonged incubation of isoproterenol-pretreated hearts with high doses of acetylcholine. In contrast, left ventricular systolic pressure was lower under basal conditions and showed a significantly higher increase during adrenergic stimulation. No changes in mRNA expression were observed. In conclusion, α7 NR has no major effect on heart rate, except when stressed hearts are exposed to a prolonged hypercholinergic state, suggesting a role in acetylcholine spillover control. In the absence of extracardiac regulatory mechanisms, left ventricular systolic impairment is revealed.

Novel single-nucleus transcriptomics unravels developmental and functionally controlled principles of mammalian neuromuscular junctions.

Prof Ohno's team (Ohkawara et al. 2023, current issue) underscored the dynamic and functional features that co-shape the embryonic and early post-natal development of mammalian neuromuscular junctions (NMJs) using single-nucleus transcriptomics which provides specific insights into the activities of individually studied nuclei and their functional characteristics. Unlike other single-nucleus transcriptomics studies, which tend to be limited to single developmental time points, this article provides novel views of the complex developmental and regulatory dynamics and embryonic cell type origins underscoring the formation of functioning mammalian NMJs by combining this transcriptomic approach with interference tests in cultured C2C12 myotubes. This reveals intriguing novel links between the particular nicotinic acetylcholine receptor genes (nAChR) and regulator transcripts thereof and enables outlining the sequential development of functioning NMJs along embryogenesis and soon after delivery. Specifically, the timewise and cell type origins of the studied nuclei emerged as essential for NMJ neurogenesis and inter-cellular transfer of specific regulators has been indicated. Breaking the barriers between distinct research subdisciplines, this study opens new neurochemistry research directions that recombine developmental, regulatory, and functional transcriptomics in NMJ-including tissues. Moreover, these findings may facilitate tests of diverse pharmaceutical and therapeutic modulators of neuromuscular functioning in health and disease, assisting the translational research progress in treating devastating neuromuscular states such as in amyotrophic lateral sclerosis, myasthenia gravis or individuals poisoned occupationally or otherwise with anticholinesterase inhibitors.

Red-on-Yellow Queen: Bio-Layer Interferometry Reveals Functional Diversity Within Micrurus Venoms and Toxin Resistance in Prey Species.

Snakes in the family Elapidae largely produce venoms rich in three-finger toxins (3FTx) that bind to the α 1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specific mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specificity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan → serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

CHRNA6 RNA In Situ Hybridization Is a Useful Tool for the Diagnosis of Extraskeletal Myxoid Chondrosarcoma.

Extraskeletal myxoid chondrosarcoma (EMC) is an uncommon mesenchymal neoplasm characteristically composed of uniform-appearing round to spindle-shaped cells with eosinophilic cytoplasm and abundant myxoid extracellular matrix. Although the majority of cases harbor a pathognomonic t(9;22) translocation that fuses EWSR1 with the orphan nuclear receptor NR4A3, there are less common variants that partner NR4A3 with TAF15, TCF12, or TFG. By immunohistochemistry, EMC has features of both cartilaginous and neuroendocrine differentiation, as evidenced by inconsistent expression of S100 protein and synaptophysin or INSM1, respectively, in a subset of cases. Given the limitations of available immunohistochemical stains for the diagnosis of EMC, we analyzed genome-wide gene expression microarray data to identify candidate biomarkers based on differential expression in EMC in comparison with other mesenchymal neoplasms. This analysis pointed to CHRNA6 as the gene with the highest relative expression in EMC (96-fold; P = 8.2 × 10-26) and the only gene with >50-fold increased expression in EMC compared with other tumors. Using RNA chromogenic in situ hybridization, we observed strong and diffuse expression of CHRNA6 in 25 cases of EMC, including both EWSR1-rearranged and TAF15-rearranged variants. All examined cases of histologic mimics were negative for CHRNA6 overexpression; however, limited CHRNA6 expression, not reaching a threshold of >5 puncta or 1 aggregate of chromogen in >25% of cells, was observed in 69 of 685 mimics (10.1%), spanning an array of mesenchymal tumors. Taken together, these findings suggest that, with careful interpretation and the use of appropriate thresholds, CHRNA6 RNA chromogenic in situ hybridization is a potentially useful ancillary histologic tool for the diagnosis of EMC.

Reconstitution of an N-AChR from Brugia malayi, an evolved change in acetylcholine receptor accessory protein requirements in filarial parasites.

Neurotransmission is an important target for anthelmintic drugs, where receptor characteristics and response can be examined through reconstitution ex vivo in Xenopus laevis oocytes. The homomeric ACR-16 nicotine sensitive acetylcholine receptors (N-AChRs) of several helminth species have been characterized in this way. Our efforts to reconstitute the N-AChR from the clade III filarial parasite, Brugia malayi using similar conditions, initially produced no detectable response. A robust response to acetylcholine is obtained from the closely related clade III parasite Ascaris suum, suggesting that specific changes have occurred between Ascaris and Brugia. N-AChRs from three species intermediate between A. suum and B. malayi were characterized to provide information on the cause. Maximal response to acetylcholine did not change abruptly, consistent with a discrete event, but rather decreased progressively from A. suum through Dracunculus medinensis, Gonglylonema pulchrum and Thelazia callipaeda. Receptor responses to the characteristic nicotine, and other agonists were generally similar. The decrease in maximal current did correlate with a delayed time to reach larger response. Together, this suggested that the failure to reconstitute the B. malayi N-AChR was one extreme of a progressive decrease and that an issue with synthesis of the receptor in oocytes was responsible. Addition of accessory proteins EMC-6, NRA-2 and NRA-4, in addition to RIC-3, produced a small, but measurable B. malayi N-AChR response. Pharmacological properties of a chimeric B. malayi N-AChR were equivalent to the other species, confirming the receptor response remains unchanged while its production is increasingly dependent on accessory proteins. One possibility is that loss of many subunits for acetylcholine receptors from the filarial nematode genome is linked to new subunit combinations that lead to such a dependence. This novel phylogenetic approach allowed the first characterization of a B. malayi AChR ex vivo and in doing so, provides a framework for the successful characterization of other receptors that have yet to be reconstituted.

Computational Design of α-Conotoxins to Target Specific Nicotinic Acetylcholine Receptor Subtypes.

Nicotinic acetylcholine receptors (nAChRs) are drug targets for neurological diseases and disorders, but selective targeting of the large number of nAChR subtypes is challenging. Marine cone snail α-conotoxins are potent blockers of nAChRs and some have been engineered to achieve subtype selectivity. This engineering effort would benefit from rapid computational methods able to predict mutational energies, but current approaches typically require high-resolution experimental structures, which are not widely available for α-conotoxin complexes. Herein, five mutational energy prediction methods were benchmarked using crystallographic and mutational data on two acetylcholine binding protein/α-conotoxin systems. Molecular models were developed for six nAChR subtypes in complex with five α-conotoxins that were studied through 150 substitutions. The best method was a combination of FoldX and molecular dynamics simulations, resulting in a predictive Matthews Correlation Coefficient (MCC) of 0.68 (85 % accuracy). Novel α-conotoxin mutants designed using this method were successfully validated by experimental assay with improved pharmaceutical properties. This work paves the way for the rapid design of subtype-specific nAChR ligands and potentially accelerated drug development.

Novel CHRNA3 variants identified in a patient with bladder dysfunction, dysautonomia, and gastrointestinal dysmotility.

Congenital anomalies of the kidney and urinary tract (CAKUT) are estimated to be responsible for 20%-50% of congenital anomalies and are also a leading etiology of early-onset renal disease. Primary CAKUT are caused by genetic factors that impair proper in-utero genitourinary tract development and secondary CAKUT result from the influence of environmental factors. The CHRNA3 gene, which encodes the Alpha-3 subunit of the nicotinic acetylcholine receptor, is hypothesized to be associated with Megacystis-microcolon-intestinal hyperperistalsis syndrome. More recently, pathogenic variants in CHRNA3 have been identified in individuals with CAKUT as well as individuals with panautonomic failure. Here we present a patient with neurogenic bladder, vesicoureteral reflux, mydriasis, and gastrointestinal dysmotility found to have novel compound heterozygous variants in CHRNA3. These findings support the consideration of CHRNA3 disruption in the differential for CAKUT with dysautonomia and gastrointestinal dysmotility.

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.