Citrobacter enshiensis sp. nov., isolated from seleniferous soil.

Two Gram-stain-negative, facultative anaerobic, rod-shaped bacterial strains, S171T and S2-9, were isolated from seleniferous soil in China. Comprehensive phylogenetic analyses based on 16S rRNA genes, multilocus sequences and whole genome sequences indicated that the two strains belonged to the genus Citrobacter. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values of strains S171T and S2-9 with the closest relative Citrobacter koseri NCTC 10786T were 83.6-83.7% and 27.7-27.8 %, respectively, which were below the species cutoff values. The ANI and dDDH values between the two strains were 97.9% and 84.8 %, respectively. The biochemical characteristics revealed that selenite tolerance, H2S and indole production, arginine dihydrolase, ornithine decarboxylase, as well as acid production from carbon sources such as d-sorbitol and arbutin are distinctive features of the two strains. Based on these results, strain S171T and strain S2-9 represent a novel species of the genus Citrobacter, for which the name Citrobacter enshiensis sp. nov. is proposed, with strain S171T (=GDMCC 1.3637T=JCM 35851T) as the type strain. The genome size of strain S171T was 4.92 Mb with a G+C content of 52.6 mol%. The genome size of strain S2-9 was 4.89 Mb with a G+C content of 52.6 mol%.

Silica nanoparticles induce ferroptosis of HUVECs by triggering NCOA4-mediated ferritinophagy.

Silica nanoparticles (SiNPs) have been widely used in electronics, chemistry, and biomedicine. Human exposure to SiNPs and possible health effects have attracted much attention. The potential cardiovascular toxicity of SiNPs and their related mechanisms are still unclear. Therefore, in this study, we investigated the toxic effects of SiNPs on human umbilical vein endothelial cells (HUVECs). We found that SiNPs could induce HUVECs ferroptosis. The results showed that the level of intracellular divalent iron and lipid peroxidation increased, and mitochondrial cristae decreased. In addition, the pretreatment of the iron chelator deferoxamine mesylate (DFO) could alleviate the ferroptosis of cells. Interestingly, pretreatment of 3-methyladenine (3-MA), an autophagy/PI3K inhibitor could partially inhibit autophagy and reduce ferroptosis, which indicated that autophagy played an important role in cell ferroptosis. Additionally, after knocking down nuclear receptor coactivator 4 (NCOA4), Ferritin Heavy Chain 1 (FTH1) expression was up-regulated, and the levels of divalent iron and lipid peroxidation decreased, which suggested that NCOA4 mediated the ferroptosis of HUVECs induced by SiNPs. In conclusion, this study shows that SiNPs can induce cardiovascular toxicity in which there is ferroptosis. NCOA4-mediated ferritinophagy and resultant ferroptosis by SiNPs may play an important role. This study provides a new theoretical strategy for the treatment and prevention of cardiovascular diseases in the future.

Flow diagram of the differential diagnosis and clinical decision making in a rare case of contrast-induced encephalopathy following cardiac catheterization: a case report.

BACKGROUND: Contrast-induced encephalopathy (CIE) is considered as an uncommon complication following cardiac catheterization. Due to the varied manifestations, CIE has no formal diagnostic criteria. In fact, the incidence of CIE may be greatly underestimated because of the difficulty in its differential diagnosis with other cerebrovascular complications. Thus, making a flow diagram according to patients' clinical symptoms and examinations after cardiac catheterization to help clinicians diagnose CIE is important and needed. CASE PRESENTATION: In this report, we describe a case of probable CIE in a 66-year-old Chinese man with hypertension who underwent cardiac catheterization with stents placement in the bifurcation lesion, during which 80 ml iopromide contrast was used. About 2 h following the procedure, the patient lost his consciousness suddenly and suffered from a status epilepticus. Malignant arrhythmias were not found through continuous electrocardiogram monitoring, but mild ST-segment elevation was displayed in leads I and aVL. The echocardiography, plasma glucose and electrolyte levels were normal. Emergency re-angiography with percutaneous transluminal coronary angioplasty was performed in the culprit lesion, which involved 60 ml iopromide contrast. However, the patient remained unconsciousness and epilepticus. Non-contrast computed tomography (CT) of the head showed cortical and subarachnoid enhancement as well as prolonged retention of contrast media in the middle cerebral artery. With supportive treatment of intravenous hydration, sedative and dehydrant, the patient recovered 3 h later and finally discharged without any neurological deficits. CONCLUSIONS: CIE is an acute reversible encephalopathy induced by contrast media. It is exceptionally challenging to make the diagnosis of CIE following cardiac catheterization since there is a lack of consensus on the definition of CIE. Via this case we reviewed the related literatures, through which a flow diagram of the differential diagnosis and clinical decision making was given, which could help to differentiate CIE from other neurological complications following cardiac catheterization.

Engineered G-Quadruplex-Embedded Self-Quenching Probes Regulate Single Probe-Based Multiplex Isothermal Amplification to Light Road Lamp Probes for Sensitized Determination of microRNAs.

Design of oligonucleotide probe-based isothermal amplification with the ability to identify miRNA biomarkers is crucial for molecular diagnostics. In this work, we engineered a miRNA-21-responsive G-quadruplex-embedded self-quenching probe (GE-SQP) that can regulate single probe-based multiplex amplifications. The free GE-SQP is tightly locked in a quenching state with no active G-quadruplexes. Introduction of target miRNA to hybridize with GE-SQP would induce a multiplex isothermal amplification to significantly build a lot of one-bulb-contained road lamp probe (OC-RLP) and two-bulb-contained road lamp probe (TC-RLP) using G-quadruplex as the lamp bulb. When lightened by thioflavin T (ThT), beams of fluorescence were emitted to show the presented miRNA-21. Specially, the whole amplification is only a one probe-involved one-step reaction without any wasted species. The mix-to-detection and all-in amplification behavior allows the sensing system a maximally maintained operation simplicity and high assay performance. In such a way, the detection range of miRNA-21 is from 1 fM to 1 nM with a limit of detection of 0.86 fM. The practicability was demonstrated by determining miRNA-21 from serum samples with acceptable results. We expect that this method can open a new avenue for exploring advanced biosensors with improved analytical performances.

Response to the Temperature and Solvent Stimulation of MOF Material in a Single-Crystal to Single-Crystal Manner.

Under solvothermal conditions, a three-dimensional mononuclear crystal AQNU-1, {[Co(H2L)(DPD)(H2O)2]·2DMA}n (H2L = 5-(bis(4-carboxybenzyl)amino)isophthalic acid, DPD = 4,4'-(2,5-diethoxy-1,4-phenylene)dipyridine) has been synthesized. The transformations of AQNU-1 to binuclear {[Co2(L)(DPD)1.5(H2O)3]·DMA·H2O}n (AQNU-2) and pentanuclear {[Co5(L)2(DPD)2(OH)2]·2H2O}n (AQNU-3) were realized by double stimulation of temperature and solvent, which were accomplished by single-crystal to single-crystal (SC-SC) reaction.

The association of chemical composition particularly the heavy metals with the oxidative potential of ambient PM2.5 in a megacity (Guangzhou) of southern China.

Atmospheric fine particulate matters (PM2.5) can cause adverse health effects through the generation of reactive oxygen species (ROS), which is normally characterized by the oxidative potential (OP). However, the particulate components that are mainly responsible for the ROS-induced OP remain controversial and warrant further investigation, especially in megacities where high exposure exists and particulate composition is complex. In this study, we measured the OP of PM2.5 using the dithiothreitol (DTT) assay with and without chelation of metals in a megacity in southern China, Guangzhou, in January and April. We explored the correlations between OP and various chemical components in PM2.5, including water-soluble ions, organic carbon (OC), elemental carbon (EC), and metal elements. There are strong correlations between OPDTTv (volume-normalized) and concentrations of PM2.5, OC, and EC, while the correlations between OPDTTm (mass-normalized) and mass-normalized water-soluble ions, OC, EC or metal elements are weak. The OP values with chelation were reduced by ∼90%, indicating that water-soluble heavy metals were the major contributors to OP of PM2.5 in Guangzhou. On the other hand, correlations between OPDTTm and OC improved significantly after the chelation of heavy metals, implying that OC explains the variance of OPDTTm although its contribution to OP is much smaller than that of heavy metals. We postulate that there might be synergetic effects between water-soluble heavy metals (which contribute most to OP) and OC (which explains the variance of OP) in ROS generation by PM2.5. The findings of the current study provide a better understanding on the critical components in PM2.5 and potential synergism that might be responsible for health effects in urban areas.

Microcrystalline silica particles induce inflammatory response via pyroptosis in primary human respiratory epithelial cells.

The mechanism of the sterile inflammatory response in the respiratory tract induced by exposure to sterile particles has not been fully elucidated. The aim of our study is to explore the earlier events in initiating inflammatory response at molecular and cellular level in primary cultured human airway epithelial cells (AEC) exposed to silica particles in order to provide information for earlier diagnosis and prevention of silica particle-induced toxicity as well as possible information on the genesis of silicosis. We isolated primary AEC from three healthy adults and treated them with silica particles at different concentrations for 48 h. We found evidence for silica-induced inflammasome activation by the co-localization of Caspase-1 and NLRP3, as well as increased levels of IL-1ß and IL-18. Lactate dehydrogenase and NucGreen analysis proved the occurrence of pyroptosis. High throughput mRNA sequencing showed that the inflammatory response and NF-κB signaling pathways were significantly enriched in gene ontology and Kyoto encyclopedia of genes and genomes analysis, and pyroptosis-related genes were up-regulated. The miR-455-3p and five lncRNAs (LOC105375913, NEAT1, LOC105375181, LOC100506098, and LOC105369370) were verified as key factors related to the mechanism by ceRNA network analysis. LOC105375913 was first discovered to be associated with inflammation in AEC. These data suggest that microcrystalline silica can induce significant inflammation and pyroptosis in human primary AEC through NLRP3 inflammasome pathway and NF-κB signaling pathway at both the gene and protein levels, and the possible mechanism could be miR-455-3p mediated ceRNA hypothesis. Our data provide a method for the studies of the respiratory toxicity of fine particulate matter and the pathogenesis of early silicosis. The miR-455-3p and five lncRNAs related ceRNA network might be the toxicity mechanism of microcrystalline silica particles to AEC.

CircPTPRA blocks the recognition of RNA N6-methyladenosine through interacting with IGF2BP1 to suppress bladder cancer progression.

BACKGROUND: Circular RNAs (circRNAs) have been found to have significant impacts on bladder cancer (BC) progression through various mechanisms. In this study, we aimed to identify novel circRNAs that regulate the function of IGF2BP1, a key m6A reader, and explore the regulatory mechanisms and clinical significances in BC. METHODS: Firstly, the clinical role of IGF2BP1 in BC was studied. Then, RNA immunoprecipitation sequencing (RIP-seq) analysis was performed to identify the circRNAs interacted with IGF2BP1 in BC cells. The overall biological roles of IGF2BP1 and the candidate circPTPRA were investigated in both BC cell lines and animal xenograft studies. Subsequently, we evaluated the regulation effects of circPTPRA on IGF2BP1 and screened out its target genes through RNA sequencing. Finally, we explored the underlying molecular mechanisms that circPTPRA might act as a blocker in recognition of m6A. RESULTS: We demonstrated that IGF2BP1 was predominantly binded with circPTPRA in the cytoplasm in BC cells. Ectopic expression of circPTPRA abolished the promotion of cell proliferation, migration and invasion of BC cells induced by IGF2BP1. Importantly, circPTPRA downregulated IGF2BP1-regulation of MYC and FSCN1 expression via interacting with IGF2BP1. Moreover, the recognition of m6A-modified RNAs mediated by IGF2BP1 was partly disturbed by circPTPRA through its interaction with KH domains of IGF2BP1. CONCLUSIONS: This study identifies exonic circular circPTPRA as a new tumor suppressor that inhibits cancer progression through endogenous blocking the recognition of IGF2BP1 to m6A-modified RNAs, indicating that circPTPRA may serve as an exploitable therapeutic target for patients with BC.

CircLIFR synergizes with MSH2 to attenuate chemoresistance via MutSα/ATM-p73 axis in bladder cancer.

BACKGROUND: Cisplatin (CDDP) has become a standard-of-care treatment for muscle-invasive bladder cancer (MIBC), while chemoresistance remains a major challenge. Accumulating evidence indicates that circular RNAs (circRNAs) are discrete functional entities. However, the regulatory functions as well as complexities of circRNAs in modulating CDDP-based chemotherapy in bladder cancer are yet to be well revealed. METHODS: Through analyzing the expression profile of circRNAs in bladder cancer tissues, RNA FISH, circRNA pull-down assay, mass spectrometry analysis and RIP, circLIFR was identified and its interaction with MSH2 was confirmed. The effects of circLIFR and MSH2 on CDDP-based chemotherapy were explored by flow cytometry and rescue experiments. Co-IP and Western blot were used to investigate the molecular mechanisms underlying the functions of circLIFR and MSH2. Biological implications of circLIFR and MSH2 in bladder cancer were implemented in tumor xenograft models and PDX models. RESULTS: CircLIFR was downregulated in bladder cancer and expression was positively correlated with favorable prognosis. Moreover, circLIFR synergizing with MSH2, which was a mediator of CDDP sensitivity in bladder cancer cells, positively modulated sensitivity to CDDP in vitro and in vivo. Mechanistically, circLIFR augmented the interaction between MutSα and ATM, ultimately contributing to stabilize p73, which triggered to apoptosis. Importantly, MIBC with high expression of circLIFR and MSH2 was more sensitive to CDDP-based chemotherapy in tumor xenograft models and PDX models. CONCLUSIONS: CircLIFR could interact with MSH2 to positively modulate CDDP-sensitivity through MutSα/ATM-p73 axis in bladder cancer. CircLIFR and MSH2 might be act as promising therapeutic targets for CDDP-resistant bladder cancer.

Organization of Protein Tyrosine Kinase-7 on Cell Membranes Characterized by Aptamer Probe-Based STORM Imaging.

Protein tyrosine kinase-7 (PTK7), as an important membrane receptor, regulates various cellular activities, including cell polarity, movement, migration, and invasion. Although lots of research studies focused on revealing its functions from the aspect of the expression of the gene and protein are present, the relationship between the spatial distribution at the single-molecule level and the function remains unclear. Through combining aptamer probe labeling and super-resolution imaging technology, after verifying the specificity and superiority of the aptamer probe, a more significant clustering distribution of PTK7 is found on the MCF10A cell basal membrane than on the apical membrane, which is thought to be related to their specific functions on different membranes. By exploring the relationship between the assembly of PTK7 and lipid rafts, actin cytoskeleton, and carbohydrate chains on the membrane, the unique distribution of PTK7 on disparate membranes is revealed to be probably because of the varied dominant position of these three factors. These findings present the detailed spatial information of PTK7 and the related potential organization mechanism on the cell membrane, which will facilitate a better understanding of the relationship between the molecular assembly and its function, as well as the overall structure of the cell membrane.

Associations between hyperhomocysteinemia and the presence and severity of acute coronary syndrome in young adults ≤ 35 years of age.

BACKGROUND: The prevalence of acute coronary syndrome (ACS) continues to increase among young Chinese adults. Homocysteine (HCY) has been suggested as a promoter of atherosclerosis leading to coronary artery disease (CAD). Yet, it remains uncertain whether HCY is associated with the ACS and the severity of coronary artery stenosis in young adults. METHODS: Young patients (18-35 years of age) diagnosed with ACS who underwent coronary angiography (CAG) at Anzhen Hospital between January 2013 and June 2019 were assigned to the ACS group. As confirmed by CAG during the same period, an equivalent age-matched population without CAD was assigned to the non-CAD group. A serum HCY level > 15 µmol/L was defined as hyperhomocysteinemia (HHCY). The Gensini score assessed the severity of coronary artery stenosis. RESULTS: A total of 1103 participants, including 828 ACS patients and 275 non-CAD subjects, were enrolled in this study. Young ACS patients had higher level of serum HCY and greater prevalence of HHCY compared with non-CAD subjects [for HCY, 16.55 (11.93-29.68) vs 12.50 (9.71-17.42), P < 0.001; for HHCY prevalence, 62.08% vs 26.18%, P < 0.001]. Multivariate logistic regression analysis with the stepwise method indicated that HHCY was an independent predictor associated with the presence of ACS, after adjusting for traditional confounders (OR, 4.561; 95% CI, 3.288-6.327; P < 0.001). Moreover, young ACS patients with HHCY had increased prevalence of ST-segment elevation myocardial infarction (STEMI) (P = 0.041), multi-vessel disease (P = 0.036), and decreased value of left ventricular ejection fraction (LVEF) (P = 0.01). Also, the HCY level was significantly correlated with Gensini Score in ACS patients (r = 0.142, P < 0.001). CONCLUSION: HHCY is significantly associated with the presence of ACS and the severity of coronary artery stenosis in young adults ≤ 35 years of age.

Combined effect of hyperhomocysteinemia and smoking on the severity of coronary artery disease in young adults ≤ 35 years of age: a hospital-based observational study.

BACKGROUND: The prevalence of coronary artery disease (CAD) continues to increase among young Chinese adults. Current smoking has been recognized as a major risk factor for premature CAD, and hyperhomocysteinaemia (HHcy) has also been suggested to be associated with CAD progression. However, the combined effect of current smoking and HHcy on the severity of coronary artery stenosis in young adults is still uncertain. METHODS: We consecutively collected young patients (18-35 years of age), diagnosed with CAD and underwent coronary angiography (CAG) at Anzhen Hospital between January 2013 and May 2020. HHcy was defined as serum homocysteine (Hcy) level > 15 µmol/L. The severity of coronary artery stenosis was evaluated by Gensini Score. The co-effect of current smoking and HHcy on CAD severity as well as the relationship between plasma Hcy, pack-years of smoking and CAD severity were assessed by multivariate linear regression analysis. RESULTS: A total of 989 participants (mean age, 33 years; 96.2% male) fulfilling the criteria were enrolled in this study. Patients with both HHcy and current smoking accounted for 39.1% of all the subjects. Multivariate liner analysis indicated both serum Hcy levels (ß 0.302; 95% CI 0.141-0.462; P < 0.001) and pack-years of smoking (ß 0.523; 95% CI 0.265-0.781; P < 0.001) were independently associated with the severity of coronary artery stenosis after adjusting for other traditional confounders. In addition, serum Hcy levels were correlated with pack-years of smoking in young CAD patients (r = 0.116, P = 0.001). Moreover, combination of HHcy and current smoking was suggested to have higher risk for CAD severity (ß 17.892; 95% CI 11.314-24.469; P < 0.001), compared with HHcy (ß 7.471; 95% CI 0.009-14.934; P = 0.048) or current smoking (ß 7.421; 95% CI 0.608-14.233; P = 0.033) alone. CONCLUSION: Combination of HHcy and smoking is independently associated with the severity of CAD in young patients ≤ 35 years of age.

Evaluation of MTBH, a novel hesperetin derivative, on the activity of hepatic cytochrome P450 isoform in vitro and in vivo using a cocktail method by HPLC-MS/MS.

1. 8-methylene-tert-butylamine-3',5,7-trihydroxy-4'-methoxyflavanone (MTBH), a novel hesperidin derivative, has potential in the prevention of hepatic disease, however, its effects on cytochrome P450 isoforms (CYP450s) remains unexplored. The purpose was to investigate the effects of MTBH on the mRNA, protein levels, and activities of six CYP450s (1A2, 2C11/9, 2D2/6, 3A1/4, 2C13/19, and 2E1) in vitro and in vivo.2. In vitro study, rat and human liver microsomes were adopted to elucidate the inhibitory effect of MTBH on six CYP450s using probe drugs. In vivo study, Sprague-Dawley male rats were treated with MTBH (25, 50, or 100 mg/kg for 28 consecutive days), phenobarbital (80 mg/kg for 12 consecutive days), or 0.5% CMC-Na solution (control group) by intragastric administration, then, the mRNA, protein levels and activities of liver CYP450s were analysed by real-time PCR, western blotting and probe-drug incubation systems, respectively.3. The in vitro study indicated that MTBH inhibits the activities of CYP3A1/4 and CYP2E1 in rat and human liver microsomes. In vivo data showed that MTBH inhibits mRNA, protein levels, and activities of CYP3A1 and CYP2E1 in medium- and high-dose MTBH groups.4. MTBH has the potential to cause drug-drug interactions when co-administered with drugs that are metabolised by CYP3A1/4 and CYP2E1.

How cognitive and reactive fear circuits optimize escape decisions in humans.

Flight initiation distance (FID), the distance at which an organism flees from an approaching threat, is an ecological metric of cost-benefit functions of escape decisions. We adapted the FID paradigm to investigate how fast- or slow-attacking "virtual predators" constrain escape decisions. We show that rapid escape decisions rely on "reactive fear" circuits in the periaqueductal gray and midcingulate cortex (MCC), while protracted escape decisions, defined by larger buffer zones, were associated with "cognitive fear" circuits, which include posterior cingulate cortex, hippocampus, and the ventromedial prefrontal cortex, circuits implicated in more complex information processing, cognitive avoidance strategies, and behavioral flexibility. Using a Bayesian decision-making model, we further show that optimization of escape decisions under rapid flight were localized to the MCC, a region involved in adaptive motor control, while the hippocampus is implicated in optimizing decisions that update and control slower escape initiation. These results demonstrate an unexplored link between defensive survival circuits and their role in adaptive escape decisions.

Quantitatively Mapping the Assembly Pattern of EpCAM on Cell Membranes with Peptide Probes.

Epithelial cell adhesion molecule (EpCAM) is an important type I transmembrane protein that is overexpressed on the surfaces of most cancer cells and involved in various biological processes such as cell adhesion and cell signaling. Although it plays crucial roles in cell functions and tumorigenesis, questions concerning the detailed morphology, molecular stoichiometry, and the assembly mechanisms of EpCAM on cell membranes have not been fully elucidated. Here, we used direct stochastic optical reconstruction microscopy (dSTORM) and relied on fluorophore-conjugated peptides to quantitatively analyze the assembly pattern of EpCAM with single-molecule precision. EpCAM was found to organize heterogeneous clusters with different sizes, which contain different numbers of EpCAM molecules on MCF-7 cell membranes. Moreover, dual-color dSTORM imaging revealed a significant correlation between EpCAM and tetraspanin CD9, and part of the EpCAM clusters could be disrupted by knockdown of CD9, which indicated that EpCAM might localize in tetraspanin-enriched microdomains (TEMs) and function cooperatively with CD9 on cell membranes. In addition, the assembly of the membrane EpCAM was found to be limited by both cytoskeleton and glycosylation. Overall, our work clarified the clustered distribution of EpCAM and revealed the potential mechanisms of its clustering at the molecular level, promoting a deeper understanding of the nano-organization of membrane proteins.

Genetic correlations among psychiatric and immune-related phenotypes based on genome-wide association data.

Individuals with psychiatric disorders have elevated rates of autoimmune comorbidity and altered immune signaling. It is unclear whether these altered immunological states have a shared genetic basis with those psychiatric disorders. The present study sought to use existing summary-level data from previous genome-wide association studies to determine if commonly varying single nucleotide polymorphisms are shared between psychiatric and immune-related phenotypes. We estimated heritability and examined pair-wise genetic correlations using the linkage disequilibrium score regression (LDSC) and heritability estimation from summary statistics methods. Using LDSC, we observed significant genetic correlations between immune-related disorders and several psychiatric disorders, including anorexia nervosa, attention deficit-hyperactivity disorder, bipolar disorder, major depression, obsessive compulsive disorder, schizophrenia, smoking behavior, and Tourette syndrome. Loci significantly mediating genetic correlations were identified for schizophrenia when analytically paired with Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus, and ulcerative colitis. We report significantly correlated loci and highlight those containing genome-wide associations and candidate genes for respective disorders. We also used the LDSC method to characterize genetic correlations among the immune-related phenotypes. We discuss our findings in the context of relevant genetic and epidemiological literature, as well as the limitations and caveats of the study.

Probing the structure of the uncoupled nicotinic acetylcholine receptor.

In the absence of activating anionic lipids and cholesterol, the nicotinic acetylcholine receptor (nAChR) from Torpedo adopts an uncoupled conformation that does not usually gate open in response to agonist. The uncoupled conformation binds both agonists and non-competitive channel blockers with a lower affinity than the desensitized state, consistent with both the extracellular agonist-binding and transmembrane channel-gating domains individually adopting resting-state like conformations. To test this hypothesis, we characterized the binding of the agonist, acetylcholine, and two fluorescent channel blockers, ethidium and crystal violet, to resting, desensitized and uncoupled nAChRs in reconstituted membranes. The measured Kd for acetylcholine binding to the uncoupled nAChR is similar to that for the resting state, confirming that the agonist binding site adopts a resting-state like conformation. Although both ethidium and crystal violet bind to the resting and desensitized channel pores with distinct affinities, no binding of either probe was detected to the uncoupled nAChR. Our data suggest that the transmembrane domain of the uncoupled nAChR adopts a conformation distinct from that of the resting and desensitized states. The lack of binding is consistent with a more constricted channel pore, possibly along the lines of what is observed in crystal structures of the prokaryotic homolog, ELIC.

Intramembrane aromatic interactions influence the lipid sensitivities of pentameric ligand-gated ion channels.

Although the Torpedo nicotinic acetylcholine receptor (nAChR) reconstituted into phosphatidylcholine (PC) membranes lacking cholesterol and anionic lipids adopts a conformation where agonist binding is uncoupled from channel gating, the underlying mechanism remains to be defined. Here, we examine the mechanism behind lipid-dependent uncoupling by comparing the propensities of two prokaryotic homologs, Gloebacter and Erwinia ligand-gated ion channel (GLIC and ELIC, respectively), to adopt a similar uncoupled conformation. Membrane-reconstituted GLIC and ELIC both exhibit folded structures in the minimal PC membranes that stabilize an uncoupled nAChR. GLIC, with a large number of aromatic interactions at the interface between the outermost transmembrane α-helix, M4, and the adjacent transmembrane α-helices, M1 and M3, retains the ability to flux cations in this uncoupling PC membrane environment. In contrast, ELIC, with a level of aromatic interactions intermediate between that of the nAChR and GLIC, does not undergo agonist-induced channel gating, although it does not exhibit the expected biophysical characteristics of the uncoupled state. Engineering new aromatic interactions at the M4-M1/M3 interface to promote effective M4 interactions with M1/M3, however, increases the stability of the transmembrane domain to restore channel function. Our data provide direct evidence that M4 interactions with M1/M3 are modulated during lipid sensing. Aromatic residues strengthen M4 interactions with M1/M3 to reduce the sensitivities of pentameric ligand-gated ion channels to their surrounding membrane environment.

Nicotinic acetylcholine receptor-lipid interactions: Mechanistic insight and biological function.

Membrane lipids are potent modulators of the nicotinic acetylcholine receptor (nAChR) from Torpedo. Lipids influence nAChR function by both conformational selection and kinetic mechanisms, stabilizing varying proportions of activatable versus non-activatable conformations, as well as influencing the transitions between these conformational states. Of note, some membranes stabilize an electrically silent uncoupled conformation that binds agonist but does not undergo agonist-induced conformational transitions. The uncoupled nAChR, however, does transition to activatable conformations in relatively thick lipid bilayers, such as those found in lipid rafts. In this review, we discuss current understanding of lipid-nAChR interactions in the context of increasingly available high resolution structural and functional data. These data highlight different sites of lipid action, including the lipid-exposed M4 transmembrane α-helix. Current evidence suggests that lipids alter nAChR function by modulating interactions between M4 and the adjacent transmembrane α-helices, M1 and M3. These interactions have also been implicated in both the folding and trafficking of nAChRs to the cell surface. We review current mechanistic understanding of lipid-nAChR interactions, and highlight potential biological roles for lipid-nAChR interactions in modulating the synaptic response. This article is part of a Special Issue entitled: Lipid-protein interactions.