Ligand-gated Ion Channel Receptors Research Articles

Structural studies of the human α1 glycine receptor via site-specific chemical cross-linking coupled with mass spectrometry

By identifying distance constraints, chemical cross-linking coupled with mass spectrometry (CX-MS) can be a powerful complementary technique to other structural methods by interrogating macromolecular protein complexes under native-like conditions. In this study, we developed a CX-MS approach to identify the sites of chemical cross-linking from a single targeted location within the human α1 glycine receptor (α1 GlyR) in its apo state. The human α1 GlyR belongs to the family of pentameric ligand-gated ion channel receptors that function in fast neurotransmission. A single chemically reactive cysteine was reintroduced into a Cys null α1 GlyR construct at position 41 within the extracellular domain of human α1 homomeric GlyR overexpressed in a baculoviral system. After purification and reconstitution into vesicles, methanethiosulfonate-benzophenone-alkyne, a heterotrifunctional cross-linker, was site specifically attached to Cys41 via disulfide bond formation. The resting receptor was then subjected to UV photocross-linking. Afterward, monomeric and oligomeric α1 GlyR bands from SDS-PAGE gels were trypsinized and analyzed by tandem MS in bottom-up studies. Dozens of intrasubunit and intersubunit sites of α1 GlyR cross-linking were differentiated and identified from single gel bands of purified protein, showing the utility of this experimental approach to identify a diverse array of distance constraints of the α1 GlyR in its resting state. These studies highlight CX-MS as an experimental approach to identify chemical cross-links within full-length integral membrane protein assemblies in a native-like lipid environment.

Serotonin Influences Insulin Secretion in Rat Insulinoma INS-1E Cells.

Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine that plays a critical role in insulin secretion, energy metabolism, and mitochondrial biogenesis. However, the action of serotonin in insulin production and secretion by pancreatic β cells has not yet been elucidated. Here, we investigated how exogenous nanomolar serotonin concentrations regulate insulin synthesis and secretion in rat insulinoma INS-1E cells. Nanomolar serotonin concentrations (10 and 50 nM) significantly increased insulin protein expression above the constant levels in untreated control cells and decreased insulin protein levels in the media. The reductions in insulin protein levels in the media may be associated with ubiquitin-mediated protein degradation. The levels of membrane vesicle trafficking-related proteins including Rab5, Rab3A, syntaxin6, clathrin, and EEA1 proteins were significantly decreased by serotonin treatment compared to the untreated control cells, whereas the expressions of Rab27A, GOPC, and p-caveolin-1 proteins were significantly reduced by serotonin treatment. In this condition, serotonin receptors, Gαq-coupled 5-HT2b receptor (Htr2b), and ligand-gated ion channel receptor Htr3a were significantly decreased by serotonin treatment. To confirm the serotonylation of Rab3A and Rab27A during insulin secretion, we investigated the protein levels of Rab3A and Rab27A, in which transglutaminase 2 (TGase2) serotonylated Rab3A but not Rab27A. The increases in ERK phosphorylation levels were consistent with increases in the expression of p-Akt. Also, the expression level of the Bcl-2 protein was significantly increased by 50 and 100 nM serotonin treatment compared to the untreated control cells, whereas the levels of Cu/Zn-SOD and Mn-SOD proteins decreased. These results indicate that nanomolar serotonin treatment regulates the insulin protein level but decreases this level in media through membrane vesicle trafficking-related proteins (Rab5, Rab3A, syntaxin6, clathrin, and EEA1), the Akt/ERK pathway, and Htr2b/Htr3a in INS-1E cells.

Protein Painting Mass Spectrometry in the Discovery of Interaction Sites within the Acetylcholine Binding Protein.

Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channel receptors that contribute to cognition, memory, and motor control in many organisms. The pharmacological targeting of these receptors, using small molecules or peptides, presents an important strategy for the development of drugs that can treat important human diseases, including neurodegenerative disorders. The Aplysia californica acetylcholine binding protein (Ac-AChBP) is a structural surrogate of the nAChR with high homology to the extracellular ligand binding domain of homopentameric nAChRs. In this study, we optimized protein-painting-based mass spectrometry to identify regions of interaction between the Ac-AChBP and several nAChR ligands. Using molecular dyes that adhere to the surface of a solubilized Ac-AChBP complex, we identified amino acid residues that constitute a contact site within the Ac-AChBP for α-bungarotoxin, choline, nicotine, and amyloid-β 1-42. By integrating innovation in protein painting mass spectrometry with computational structural modeling, we present a new experimental tool for analyzing protein interactions of the nAChR.

Role of β3 subunit of the GABA type A receptor in triple negative breast cancer proliferation, migration, and cell cycle progression

ABSTRACT Triple negative breast cancer (TNBC) is known for its heterogeneous nature and aggressive onset. The unresponsiveness to hormone therapies and immunotherapy and the toxicity of chemotherapeutics account for the limited treatment options for TNBC. Ion channels have emerged as possible therapeutic candidates for cancer therapy, but little is known about how ligand gated ion channels, specifically, GABA type A ligand-gated ion channel receptors (GABAAR), affect cancer pathogenesis. Our results show that the GABAA β3 subunit is expressed at higher levels in TNBC cell lines than non-tumorigenic cells, therefore contributing to the idea that limiting the GABAAR via knockdown of the GABAA β3 subunit is a potential strategy for decreasing the proliferation and migration of TNBC cells. We employed pharmacological and genetic approaches to investigate the role of the GABAA β3 subunit in TNBC proliferation, migration, and cell cycle progression. The results suggest that pharmacological antagonism or genetic knockdown of GABAA β3 subunit decreases TNBC proliferation and migration. In addition, GABAA β3 subunit knockdown causes cell cycle arrest in TNBC cell lines via decreased cyclin D1 and increased p21 expression. Our findings suggest that membrane bound GABAA receptors containing the β3 subunit can be further developed as a potential novel target for the treatment of TNBC.

Progress on functions of intracellular domain of trimeric ligand-gated ion channels.

Ligand-gated ion channels are a large category of essential ion channels, modulating their state by binding to specific ligands to allow ions to pass through the cell membrane. Purinergic ligand-gated ion channel receptors (P2XRs) and acid-sensitive ion channels (ASICs) are representative members of trimeric ligand-gated ion channel. Recent studies have shown that structural differences in the intracellular domain of P2XRs may determine the desensitization process. The lateral fenestrations of P2XRs potentially serve as a pathway for ion conductance and play a decisive role in ion selectivity. Phosphorylation of numerous amino acid residues in the P2XRs are involved in regulating the activity of ion channels. Additionally, the P2XRs interact with other ligand-gated ion channels including N-methyl-D-aspartate receptors, γ-aminobutyric acid receptors, 5-hydroxytryptamin receptors and nicotinic acetylcholine receptors, mediating physiological processes such as synaptic plasticity. Conformational changes in the intracellular domain of the ASICs expose binding sites of intracellular signal partners, facilitating metabolic signal transduction. Amino acids such as Val16, Ser17, Ile18, Gln19 and Ala20 in the ASICs participate in channel opening and membrane expression. ASICs can also bind to intracellular proteins, such as CIPP and p11, to regulate channel function. Many phosphorylation sites at the C-terminus and N-terminus of ASICs are involved in the regulation of receptors. Furthermore, ASICs are involved in various physiological and pathophysiological processes, which include pain, ischemic stroke, psychiatric disorders, and neurodegenerative disease. In this article, we review the roles of the intracellular domains of these trimeric ligand-gated ion channels in channel gating as well as their physiological and pathological functions, in order to provide new insights into the discovery of related drugs.

Understanding the GABAA Receptor: Implications for Anesthesia and Beyond

AbstractGamma-aminobutyric acid (GABA), a nonpeptide amino acid transmitter, is a major component of modern neuropharmacology and one of the most crucial target sites for general anesthetics and therapeutic drugs. GABA type A receptors (GABAARs) are the most abundant inhibitory neurotransmitter receptors in the central nervous system. They are part of the rapid-acting, ligand-gated ion channel (LGIC) receptor category, a pentameric Cys-loop superfamily member that mediates inhibitory neurotransmission in the mature brain. GABAARs mainly consist of two α subunits, two β subunits, and one additional subunit from either γ or δ arranged around a central chloride (Cl-) selective channel. Multiple GABAAR subunit subtypes and splice variants have been identified. Each variant of GABAAR exhibits distinct biophysical and pharmacologic properties. Several compounds allosterically modulate the GABAAR positively or negatively. The widely used positive GABAAR modulators include benzodiazepines (anxiolytic and anticonvulsant), general anesthetics (volatile agents like isoflurane, and intravenous agents like barbiturates, etomidate, and propofol), long-chain alcohols, some anticonvulsants, and neuroactive steroids. The binding sites for each drug are distinctly different. The anesthetic drugs enhance receptor-mediated synaptic transmission and thus interrupt the thalamocortical transmission, which controls the sleep–wake patterns. Abnormality in the GABAAR function has been implicated in several neurological conditions, such as sleep disorders, seizures, depression, cognitive function, neurological recovery after injury, and neuroplasticity. Understanding the GABAAR lays the foundation for the development of highly specific drugs in the treatment of neurological disorders and general anesthesia.

Effects of RIC-3 on the expression profile of serotonin type 3A receptors.

Serotonin type 3A (5-HT3A) receptors belong to the Cys-loop ligand-gated ion channel receptor superfamily. 5-HT3A receptors are found in high densities in certain brain regions, have an involvement with brain-gut signaling circuitry and other non-serotonergic synaptic activities, which make them effective and potential therapeutic targets for treatments of many conditions such as irritable bowel syndrome, chemotherapy-induced vomiting, inflammation, and psychiatric disorders. RIC-3, a transmembrane protein and endoplasmic reticulum resident chaperone, acts as a molecular chaperone of cation-conducting Cys-loop receptors, both serotonin type 3A (5-HT3A) and nicotinic acetylcholine receptors. RIC-3 is needed for efficient receptor folding, assembly and trafficking/functional surface expression. RIC-3 interacts directly with the intracellular domain of serotonin type 3A subunits (ICD). RIC-3 has two binding sites with a shared duplicated motif in 5HT3A subunits, one in the MX-helix and one in the MAM4-helix. Toward drug discovery based on protein-protein interactions, we here aimed to determine the effect of RIC-3 on the expression profile of serotonin type 3A receptors. For this study, 5-HT3A constructs were genetically engineered, and the functional surface expression was examined using two-electrode voltage clamp and immunoblot analysis/western blot. We found that when 5-HT3A channels were co-injected with RIC-3, RIC-3 inhibits 5-HT3A current amplitudes elicited by serotonin in Xenopus oocytes; Ala replacements at residues W347, R349, and L353 (MX) or residues W447, R449, and L454 (MAM4) on the ICD weakened the inhibitory effect of RIC-3 on the functional surface expression of the channels. We infer that in Xenopus oocytes, RIC-3 reduction of functional surface expression of 5HT3A receptors is mediated by binding to the two identified binding sites.

Purinergic signaling: Diverse effects and therapeutic potential in cancer.

Regardless of improved biological insights and therapeutic advances, cancer is consuming multiple lives worldwide. Cancer is a complex disease with diverse cellular, metabolic, and physiological parameters as its hallmarks. This instigates a need to uncover the latest therapeutic targets to advance the treatment of cancer patients. Purines are building blocks of nucleic acids but also function as metabolic intermediates and messengers, as part of a signaling pathway known as purinergic signaling. Purinergic signaling comprises primarily adenosine triphosphate (ATP) and adenosine (ADO), their analogous membrane receptors, and a set of ectonucleotidases, and has both short- and long-term (trophic) effects. Cells release ATP and ADO to modulate cellular function in an autocrine or paracrine manner by activating membrane-localized purinergic receptors (purinoceptors, P1 and P2). P1 receptors are selective for ADO and have four recognized subtypes-A1, A2A, A2B, and A3. Purines and pyrimidines activate P2 receptors, and the P2X subtype is ligand-gated ion channel receptors. P2X has seven subtypes (P2X1-7) and forms homo- and heterotrimers. The P2Y subtype is a G protein-coupled receptor with eight subtypes (P2Y1/2/4/6/11/12/13/14). ATP, its derivatives, and purinoceptors are widely distributed in all cell types for cellular communication, and any imbalance compromises the homeostasis of the cell. Neurotransmission, neuromodulation, and secretion employ fast purinergic signaling, while trophic purinergic signaling regulates cell metabolism, proliferation, differentiation, survival, migration, invasion, and immune response during tumor progression. Thus, purinergic signaling is a prospective therapeutic target in cancer and therapy resistance.

P2-type purinergic signaling in the regulation of pancreatic β-cell functional plasticity as a promising novel therapeutic approach for the treatment of type 2 diabetes?

Diabetes Mellitus is a metabolic disorder characterized by a chronic hyperglycemia due to an impaired insulin secretion and a decreased in peripheral insulin sensitivity. This disease is a major public health problem due to it sharp prevalence. Therefore, it is crucial to readapt therapeutic approaches for the treatment of this pathology. One of the strategies would be through P2-type purinergic receptors pathway via ATP binding. In addition to its well-known role as an intracellular energy intermediary in numerous biochemical and physiological processes, ATP is also an important extracellular signaling molecule. ATP mediates its effects by binding and activating two classes of P2 purinoreceptors: P2X receptors that are ligand-gated ion channel receptors, existing in seven isoforms (P2X 1 to 7) and P2Y receptors that are G-protein coupled receptors, existing in eight isoforms (P2Y 1/2/4/6/11/12/13/14). These receptors are ubiquitously distributed and involved in numerous physiological processes in several tissues. The concept of purinergic signaling, originally formulated by Geoffrey Burnstock (1929-2020), was also found to mediate various responses in the pancreas. Several studies have shown that P2 receptors are expressed in the endocrine pancreas, notably in β cells, where ATP could modulate their function but also their plasticity and thus play a physiological role in stimulating insulin secretion to face some metabolic demands. In this review, we provide a historical perspective and summarize current knowledge on P2-type purinergic signaling in the regulation of pancreatic β-cell functional plasticity, which would be a promising novel therapeutic approach for the treatment of type 2 diabetes.

Regulation of Inhibitory Signaling at the Receptor and Cellular Level; Advances in Our Understanding of GABAergic Neurotransmission and the Mechanisms by Which It Is Disrupted in Epilepsy.

Inhibitory signaling in the brain organizes the neural circuits that orchestrate how living creatures interact with the world around them and how they build representations of objects and ideas. Without tight control at multiple points of cellular engagement, the brain’s inhibitory systems would run down and the ability to extract meaningful information from excitatory events would be lost leaving behind a system vulnerable to seizures and to cognitive decline. In this review, we will cover many of the salient features that have emerged regarding the dynamic regulation of inhibitory signaling seen through the lens of cell biology with an emphasis on the major building blocks, the ligand-gated ion channel receptors that are the first transduction point when the neurotransmitter GABA is released into the synapse. Epilepsy association will be used to indicate importance of key proteins and their pathways to brain function and to introduce novel areas for therapeutic intervention.

Local modulation by presynaptic receptors controls neuronal communication and behaviour.

Central nervous system neurons communicate via fast synaptic transmission mediated by ligand-gated ion channel (LGIC) receptors and slower neuromodulation mediated by G protein-coupled receptors (GPCRs). These receptors influence many neuronal functions, including presynaptic neurotransmitter release. Presynaptic LGIC and GPCR activation by locally released neurotransmitters influences neuronal communication in ways that modify effects of somatic action potentials. Although much is known about presynaptic receptors and their mechanisms of action, less is known about when and where these receptor actions alter release, especially in vivo. This Review focuses on emerging evidence for important local presynaptic receptor actions and ideas for future studies in this area.

The ligand-gated ion channel P2X7 receptor mediates NLRP3/caspase-1-mediated pyroptosis in cerebral cortical neurons of juvenile rats with sepsis

Sepsis-associated encephalopathy (SAE) is a common complication of severe sepsis. Some studies have suggested that P2X7 receptors, a ligand-gated ion channel receptor subgroup activated by extracellular ATP, plays an important role in cell pyroptosis. However, the role of P2X7 receptors in the development of SAE with pyroptosis and its pathways are still unclear. In this study, we established a juvenile rat model of sepsis by cecal ligation and perforation, and showed that there was a significant increase in P2X7 receptor expression in the cortex of juvenile rats with sepsis. When the P2X7 receptor antagonist was administrated, the pyroptosis and extracellular-signal-regulated kinase (ERK) 1/2 signaling pathway were inhibited, and when the P2X7 receptor agonist was administrated, the pyroptosis and ERK1/2 signaling pathway were further activated. In addition, we also found that the administration of ERK1/2 signaling pathway inhibitor not only weakened downstream pyroptosis, but also caused the inhibition of upstream P2X7 receptor expression. In conclusion, our findings illustrated that the ligand-gated ion channel P2X7 receptor mediates NLRP3/caspase-1-related pyroptosis in cerebral cortex of juvenile rats with sepsis through ERK1/2 signaling pathway and plays a neuroprotective role.

Role of purines in regulation of metabolic reprogramming

Purines, among most influential molecules, are reported to have essential biological function by regulating various cell types. A large number of studies have led to the discovery of many biological functions of the purine nucleotides such as ATP, ADP, and adenosine, as signaling molecules that engage G protein-coupled or ligand-gated ion channel receptors. The role of purines in the regulation of cellular functions at the gene or protein level has been well documented. With the advances in multiomics, including those from metabolomic and bioinformatic analyses, metabolic reprogramming was identified as a key mechanism involved in the regulation of cellular function under physiological or pathological conditions. Recent studies suggest that purines or purine-derived products contribute to important regulatory functions in many fundamental biological and pathological processes related to metabolic reprogramming. Therefore, this review summarizes the role and potential mechanism of purines in the regulation of metabolic reprogramming. In particular, the molecular mechanisms of extracellular purine- and intracellular purine-mediated metabolic regulation in various cells during disease development are discussed. In summary, our review provides an extensive resource for studying the regulatory role of purines in metabolic reprogramming and sheds light on the utilization of the corresponding peptides or proteins for disease diagnosis and therapy.

Methylosome protein 50 associates with the purinergic receptor P2X5 and is involved in osteoclast maturation.

Purinergic signaling plays important roles in bone. P2X5, a member of ligand-gated ion channel receptors, has been demonstrated to regulate osteoclast maturation. However, the molecular mechanism of P2X5-mediated osteoclast regulation remains unclear. Here, we identified methylosome protein 50 (MEP50), a critical cofactor of the protein arginine methyltransferase 5 (PRMT5), as a P2X5-associating molecule. RNAi-mediated knockdown of MEP50 results in decreased formation of mature osteoclasts. MEP50 associates with P2X5, and this association requires the C-terminal intracellular region of P2X5. Additionally, impaired maturation of P2X5-deficient osteoclasts could be restored by transduction of full-length P2X5, but not a C-terminal deletion mutant of P2X5. These results indicate that P2X5 associates with MEP50 and suggest a link between the PRMT5 complex and P2X5 signaling in osteoclast maturation.

Cytisine - From the Past to the Future.

Neuronal nicotinic acetylcholine receptors are ligand-gated ion channel receptors, distributed throughout central nervous system, as well as in peripheral ganglia and some non-neuronal cells. Cytisine, a qulinolizidine alkaloid, could be considered a high affinity ligand of those receptors. It is a partial agonist of β2*-containing receptors and a full agonist of α7 and β4*-containing receptors. At present, pharmacodynamic properties of cytisine are leveraged only in a few European countries where it is available as medicinal product (Desmoxan and Tabex) indicated in the pharmacotherapy of nicotine addiction. Cytisine mimics the influence of nicotine on α4β2* receptors, but with higher affinity and lower activity. It lowers rewarding and reinforcing effects of nicotine in smoking persons and reduces withdrawal symptoms and craving in quitting ones. The results of non-clinical studies suggest that cytisine could affect ethanol consumption, has an antidepressant and neuroprotective effect and could be useful in reducing body mass and preventing weight gain. Although there is a lack of research on cytisine in the treatment of areca nuts usage, the preliminary data suggest its usefulness. The combination of cytisine and Trolox C was selected as a possible effective treatment for type 2 diabetes. Though these drugs alone are not effective, their theoretical usefulness was confirmed in animal models. Treatment with cytisine is an effective, cost-efficient, affordable and well tolerated nicotine addiction therapy. Potential new indications for cytisine include the treatment of alcoholism, areca nuts usage, Parkinson's disease, an autonomic-system failure. Further studies are necessary.

Effects of Overexpressed P2X7 on Leukemia Progression in Mouse Acute Myeloid Leukemia Model

P2X7 is one of the P2X family ligand-gated ion channel receptors. High level expression of P2X7 is reported in various malignant cells. High level expression of P2X7 was also detected in leukemia patients, especially in relapsed cases. A hyposensitive P2X7 mutant, N187D P2X7, was cloned from leukemia cells. However, the role of P2X7 on the progression of leukemia was poorly understood. In the present study, we studied the effects of P2X7 receptor in acute myeloid leukemia mouse model.We established MLL-AF9 induced AML mouse model with high level expression of P2X7 (MA9-P2X7). High level of P2X7 significantly accelerated the progression of leukemia. Homing capability experiment demonstrated that MA9-P2X7 cells had lower homing potential. The apoptotic rate of freshly isolated leukemia cells had no significant difference between two groups. However, both in vitro culture experiments and in vivo BrdU incorporation assay demonstrated that MA9-P2X7 cells had enhanced proliferation potential, i.e. more S and G2/M phase cells but less G0/G1 phase cells. Moreover, upon treatment with Ara-C, though MA9-P2X7 cells were more sensitive to Ara-C, but the mice had shorter survival time. Furthermore, in vitro colony assay and limiting dilution transplantation experiments showed that MA9-P2X7 cells formed more colonies and had a 7.25-folds increase in LICs frequency. We also detected the expression of c-Kit, and the results showed that the majority of MA9-P2X7 cells were c-Kit+, whereas control cells have two populations and more than half of them were c-Kit-. Leukemia mice in MA9-P2X7 group had shorter survival time than those in the c-Kit+ control group.Our results suggested that leukemia cells overexpressing P2X7 possessed the characteristics of both greater proliferation potential and higher LICs frequency, which contributed to the accelerated progression of leukemia.This work was supported by grants 81570153, 81770183 and 81170511 from the National Natural Science Foundation of China (NSFC); programs 2016-I2M-2-006 and 2017-I2M-1-015 from the CAMS Innovation Fund for Medical Sciences (CIFMS); grant 17JCZDJC35000 from the Tianjin Natural Science Foundation; and Graduate Student Innovation Fund (2014-0710-1021) from Peking Union Medical College. Z.GG. is a recipient of the New Century Excellent Talents in University (NCET-08-0329). DisclosuresNo relevant conflicts of interest to declare.

Genome-wide identification, characterization and classification of ionotropic glutamate receptor genes (iGluRs) in the malaria vector Anopheles sinensis (Diptera: Culicidae)

BackgroundIonotropic glutamate receptors (iGluRs) are conserved ligand-gated ion channel receptors, and ionotropic receptors (IRs) were revealed as a new family of iGluRs. Their subdivision was unsettled, and their characteristics are little known. Anopheles sinensis is a major malaria vector in eastern Asia, and its genome was recently well sequenced and annotated.MethodsWe identified iGluR genes in the An. sinensis genome, analyzed their characteristics including gene structure, genome distribution, domains and specific sites by bioinformatic methods, and deduced phylogenetic relationships of all iGluRs in An. sinensis, Anopheles gambiae and Drosophila melanogaster. Based on the characteristics and phylogenetics, we generated the classification of iGluRs, and comparatively analyzed the intron number and selective pressure of three iGluRs subdivisions, iGluR group, Antenna IR and Divergent IR subfamily.ResultsA total of 56 iGluR genes were identified and named in the whole-genome of An. sinensis. These genes were located on 18 scaffolds, and 31 of them (29 being IRs) are distributed into 10 clusters that are suggested to form mainly from recent gene duplication. These iGluRs can be divided into four groups: NMDA, non-NMDA, Antenna IR and Divergent IR based on feature comparison and phylogenetic analysis. IR8a and IR25a were suggested to be monophyletic, named as Putative in the study, and moved from the Antenna subfamily in the IR family to the non-NMDA group as a sister of traditional non-NMDA. The generated iGluRs of genes (including NMDA and regenerated non-NMDA) are relatively conserved, and have a more complicated gene structure, smaller ω values and some specific functional sites. The iGluR genes in An. sinensis, An. gambiae and D. melanogaster have amino-terminal domain (ATD), ligand binding domain (LBD) and Lig_Chan domains, except for IR8a that only has the LBD and Lig_Chan domains. However, the new concept IR family of genes (including regenerated Antenna IR, and Divergent IR), especially for Divergent IR are more variable, have a simpler gene structure (intron loss phenomenon) and larger ω values, and lack specific functional sites. These IR genes have no other domains except for Antenna IRs that only have the Lig_Chan domain.ConclusionsThis study provides a comprehensive information framework for iGluR genes in An. sinensis, and generated the classification of iGluRs by feature and bioinformatics analyses. The work lays the foundation for further functional study of these genes.

Purine and purinergic receptors.

Adenosine 5′-triphosphate acts as an extracellular signalling molecule (purinergic signalling), as well as an intracellular energy source. Adenosine 5′-triphosphate receptors have been cloned and characterised. P1 receptors are selective for adenosine, a breakdown product of adenosine 5′-triphosphate after degradation by ectonucleotidases. Four subtypes are recognised, A1, A2A, A2B and A3 receptors. P2 receptors are activated by purine and by pyrimidine nucleotides. P2X receptors are ligand-gated ion channel receptors (seven subunits (P2X1-7)), which form trimers as both homomultimers and heteromultimers. P2Y receptors are G protein-coupled receptors (eight subtypes (P2Y1/2/4/6/11/12/13/14)). There is both purinergic short-term signalling and long-term (trophic) signalling. The cloning of P2X-like receptors in primitive invertebrates suggests that adenosine 5′-triphosphate is an early evolutionary extracellular signalling molecule. Selective purinoceptor agonists and antagonists with therapeutic potential have been developed for a wide range of diseases, including thrombosis and stroke, dry eye, atherosclerosis, kidney failure, osteoporosis, bladder incontinence, colitis, neurodegenerative diseases and cancer.

P2X7 receptor large pore signaling in avian Müller glial cells.

ATP is a pleiotropic molecule that promotes extra- and intracellular signaling to regulate numerous functions. This nucleotide activates purine and pyrimidine receptors at the plasma membrane, categorized as ionotropic P2X or G-protein-coupled receptor (GPCR) P2Y receptors. P2X are ligand-gated ion channel receptors, expressed in both retinal neurons and Müller cells leading to neuron-glia communication, calcium waves and neurovascular coupling. However, how P2X pore is formed upon ATP activation and how signaling pathways regulates the complex is still a matter of controversy. Here we studied the properties of the P2X7 receptor (P2X7R) using electrophysiology, single cell Ca2+ imaging, and dye uptake assay in purified avian Müller glia in culture. Our data show that ATP (or benzoyl-benzoyl ATP, BzATP) evoked large inward currents in patch-clamp studies while addition of P2X7R antagonist such as brilliant Blue G (BBG), abolished these currents. Ruthenium red (RU-2), a general transient receptor potential (TRP) inhibitor, reduced currents induced by ATP. Our data also point to the involvement of mitogen activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), Ca2+-calmodulin kinase II (CAMKII), microtubules or protein kinase C (PKC) modulating ATP-induced ionic current in Müller cells. We show that ATP induced Ca2+ influx, partially inhibited by P2X7R antagonists (oxidized ATP or BBG), and totally inhibited by blockers of other pores such as transient receptor potential (TRPs) or connexin hemichannel. Additionally, MAPK, PKC, PI3K or CAMKII inhibitors also are involved in the modulation of intracellular calcium signaling. Finally, ATP induced 80-90% of dye uptake in Muller glia cells, while oxidized ATP (oATP), BBG or A740003 inhibited this effect. We conclude that large conductance channel and other P2XRs are not involved in the ATP-induced dye uptake, but signaling pathways such as MAPK, PI3-K, microtubules or PKC are involved in pore formation.

Mechanism of partial agonism in AMPA-type glutamate receptors

Neurotransmitters trigger synaptic currents by activating ligand-gated ion channel receptors. Whereas most neurotransmitters are efficacious agonists, molecules that activate receptors more weakly—partial agonists—also exist. Whether these partial agonists have weak activity because they stabilize less active forms, sustain active states for a lesser fraction of the time or both, remains an open question. Here we describe the crystal structure of an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) ligand binding domain (LBD) tetramer in complex with the partial agonist 5-fluorowillardiine (FW). We validate this structure, and others of different geometry, using engineered intersubunit bridges. We establish an inverse relation between the efficacy of an agonist and its promiscuity to drive the LBD layer into different conformations. These results suggest that partial agonists of the AMPAR are weak activators of the receptor because they stabilize multiple non-conducting conformations, indicating that agonism is a function of both the space and time domains.