Ligand-gated Ion Channel Receptors Research Articles

P2X7 receptor stimulates breast cancer cell invasion and migration via the AKT pathway.

Purinergic signaling has been implicated in the regulation of many cellular processes. A high concentration of ATP has been observed in the tumor microenvironment, suggesting a possible role of extracellular ATP in tumor progression. The P2X7 receptor, which belongs to the ligand-gated ion channel receptor family, is involved in tumor development and metastasis. In the present study, we found that extracellular ATP stimulated the invasion and migration of human T47D breast cancer cells, in a dose-dependent manner. BzATP (ATP analogue), but not ADP, also promoted invasion and migration. We further found that the P2X7 receptor was highly expressed in the T47D cells. After knockdown of the P2X7 receptor, ATP-stimulated invasion and migration were markedly inhibited. Moreover, activation of the P2X7 receptor by ATP downregulated the protein level of E-cadherin and upregulated the production of MMP-13. In addition, ATP time-dependently induced the activation of AKT via the P2X7 receptor, and the AKT pathway was required for the ATP-mediated invasion and migration. Taken together, our results revealed that activation of the P2X7 receptor by ATP promotes breast cancer cell invasion and migration, possibly via activation of the AKT pathway and regulation of E-cadherin and MMP-13 expression. Therefore, the P2X7 receptor may be a useful therapeutic target for the treatment of breast cancer.

Establishment of a Long-Term Chick Forebrain Neuronal Culture on a Microelectrode Array Platform.

The biosensor system formed by culturing primary animal neurons on a microelectrode array (MEA) platform is drawing an increasing research interest for its power as a rapid, sensitive, functional neurotoxicity assessment, as well as for many other electrophysiological related research purposes. In this paper, we established a long-term chick forebrain neuron culture (C-FBN-C) on MEAs with a more than 5 month long lifespan and up to 5 month long stability in morphology and physiological function; characterized the C-FBN-C morphologically, functionally, and developmentally; partially compared its functional features with rodent counterpart; and discussed its pros and cons as a novel biosensor system in comparison to rodent counterpart and human induced pluripotent stem cells (hiPSCs). Our results show that C-FBN-C on MEA platform 1) can be used as a biosensor of its own type in a wide spectrum of basic biomedical research; 2) is of value in comparative physiology in cross-species studies; and 3) may have potential to be used as an alternative, cost-effective approach to rodent counterpart within shared common functional domains (such as specific types of ligand-gated ion channel receptors and subtypes expressed in the cortical tissues of both species) in large-scale environmental neurotoxicant screening that would otherwise require millions of animals.

New Hyperekplexia Mutations Provide Insight into Glycine Receptor Assembly, Trafficking, and Activation Mechanisms

Hyperekplexia is a syndrome of readily provoked startle responses, alongside episodic and generalized hypertonia, that presents within the first month of life. Inhibitory glycine receptors are pentameric ligand-gated ion channels with a definitive and clinically well stratified linkage to hyperekplexia. Most hyperekplexia cases are caused by mutations in the α1 subunit of the human glycine receptor (hGlyR) gene (GLRA1). Here we analyzed 68 new unrelated hyperekplexia probands for GLRA1 mutations and identified 19 mutations, of which 9 were novel. Electrophysiological analysis demonstrated that the dominant mutations p.Q226E, p.V280M, and p.R414H induced spontaneous channel activity, indicating that this is a recurring mechanism in hGlyR pathophysiology. p.Q226E, at the top of TM1, most likely induced tonic activation via an enhanced electrostatic attraction to p.R271 at the top of TM2, suggesting a structural mechanism for channel activation. Receptors incorporating p.P230S (which is heterozygous with p.R65W) desensitized much faster than wild type receptors and represent a new TM1 site capable of modulating desensitization. The recessive mutations p.R72C, p.R218W, p.L291P, p.D388A, and p.E375X precluded cell surface expression unless co-expressed with α1 wild type subunits. The recessive p.E375X mutation resulted in subunit truncation upstream of the TM4 domain. Surprisingly, on the basis of three independent assays, we were able to infer that p.E375X truncated subunits are incorporated into functional hGlyRs together with unmutated α1 or α1 plus β subunits. These aberrant receptors exhibit significantly reduced glycine sensitivity. To our knowledge, this is the first suggestion that subunits lacking TM4 domains might be incorporated into functional pentameric ligand-gated ion channel receptors.

Modeling complex biological systems: From solution chemistry to membranes and channels

Complex biological systems are intimately linked to their environment, a very crowded and equally complex solution compartmentalized by fluid membranes. Modeling such systems remains challenging and requires a suitable representation of these solutions and their interfaces. Here, we focus on particle-based modeling at an atomistic level using molecular dynamics (MD) simulations. As an example, we discuss important steps in modeling the solution chemistry of an ion channel of the ligand-gated ion channel receptor family, a major target of many drugs including anesthetics and addiction treatments. The bacterial pentameric ligand-gated ion channel (pLGIC) called GLIC provides clues about the functional importance of solvation, in particular for mechanisms such as permeation and gating. We present some current challenges along with promising novel modeling approaches.

An outline of desensitization in pentameric ligand-gated ion channel receptors.

Pentameric ligand-gated ion channel (pLGIC) receptors exhibit desensitization, the progressive reduction in ionic flux in the prolonged presence of agonist. Despite its pathophysiological importance and the fact that it was first described over half a century ago, surprisingly little is known about the structural basis of desensitization in this receptor family. Here, we explain how desensitization is defined using functional criteria. We then review recent progress into reconciling the structural and functional basis of this phenomenon. The extracellular-transmembrane domain interface is a key locus. Activation is well known to involve conformational changes at this interface, and several lines of evidence suggest that desensitization involves a distinct conformational change here that is incompatible with activation. However, major questions remain unresolved, including the structural basis of the desensitization-induced agonist affinity increase and the mechanism of pore closure during desensitization.

The Cys-loop pentameric ligand-gated ion channel receptors: 50 years on

This year, 2011, the Department of Pharmacology at the University of Alberta celebrated its 50th anniversary. This timeframe covers nearly the entire history of Cys-loop pentameric ligand-gated ion channel (pLGIC) research. In this review we consider how major technological advancements affected our current understanding of pLGICs, and highlight the contributions made by members of our department. The individual at the center of our story is Susan Dunn; her passing earlier this year has robbed the Department of Pharmacology and the research community of a most insightful colleague. Her dissection of ligand interactions with the nAChR, together with their interpretation, was the hallmark of her extensive collaborations with Michael Raftery. Here, we highlight some electrophysiological studies from her laboratory over the last few years, using the technique that she introduced to the department in Edmonton, the 2-electrode voltage-clamp of Xenopus oocytes. Finally, we discuss some single-channel studies of the anionic GlyR and GABA(A)R that prefaced the introduction of this technique to her laboratory.

A Salt Bridge Crucial for Glycine Receptor Function

The glycine receptor (GlyR) chloride channel is a member of the pentameric ligand-gated ion channel (pLGIC) receptor family. These channels mediate fast inhibitory transmission in the nervous system. Recent structural data and a wide body of functional data have underlined the importance of the interface between the ligand-binding domain and the transmembrane domain in channel gating. A number of charged residues at this interface have been shown to form salt bridges critical for channel function in other pLGICs. However, no such interaction has been demonstrated in the GlyR. Mutations at D148 (Cys-loop) and R218 (pre-M1) in the homomeric alpha 1 GlyR can have drastic effects on GlyR function and the single mutants D148C and R218C failed to produce glycine-induced currents in Xenopus oocytes. However, the D148C, R218C double mutant resulted in receptors that produced glycine-gated currents with only a small decrease in glycine sensitivity compared to WT. Importantly, sub-saturating glycine-evoked currents were dramatically increased by the reducing agent dithiothreitol. This suggests that D148C and R218C can form a disulphide bridge, possibly indicating an electrostatic interaction between D148 and R218 that stabilizes the channel closed state. The possibilty of a state-dependent salt bridge between D148 and R218 raised the question how the charges are stabilised in the open state. Interestingly, structural studies have suggested that R218 is flanked by two highly conserved phenylalanine side chains. To test, if these aromatic amino acids interact with R218 electrostically via a cation-pi interaction, we incorporated a series of fluorinated Phe derivatives using the in vivo non-sense suppression method. However, the fluorination-induced changes in the glycine sensitivity were not consistent with a cation-pi interaction. Taken together, these results point towards a crucial salt bridge between D148 and R218 but no electrostatic contributions by the flanking phenylalanines.

Characterizing Ligand-Gated Ion Channel Receptors with Genetically Encoded Ca++ Sensors

We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC) by developing sensor cells stably expressing a Ca2+ permeable LGIC and a genetically encoded Förster (or fluorescence) resonance energy transfer (FRET)-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT3A serotonin receptors and a chimera of human α7/mouse 5-HT3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.

P2X7-mediated chemoprevention of epithelial cancers

Background: Anti-apoptotic mechanisms contribute to the development of cancer. The purinergic ligand-gated ion channel receptor P2X7 system is an important pro-apoptosis modulator in epithelial cells, and augmentation of P2X7-mediated apoptosis has been proposed as a novel pharmacological modality for chemoprevention and treatment of epithelial cancers. Objective: This review focuses on the progress towards clinical application of therapies that directly modulate P2X7-mediated apoptosis. This paper reviews and discusses P2X7-mediated apoptosis in the context of prevention and growth-control of epithelial cancer cells, and summarizes recent data in the mouse skin model in vivo. Methods: Data for this review were identified from the PubMed and MEDLINE databases. Summary/conclusions: The growing understanding of mechanisms of P2X7-mediated apoptosis has generated a novel strategy for targeting directly and specifically skin neoplasia. The data link directly, for the first time, upregulation of apoptosis with cancer chemoprevention. Striking antitumor efficacy has been achieved in the rodent cancer model, and it is likely that the recently gained wealth of knowledge about the mechanisms of P2X7-control of apoptosis will eventually be translated into new clinical therapies for epithelial cancers.

A comprehensive study on the 5-hydroxytryptamine 3A receptor binding of agonists serotonin and m-chlorophenylbiguanidine

Serotonin type 3 receptors (5-HT 3R) are members of the ligand gated ion channel receptor family. In this study, the interactions of the agonists serotonin (5-HT) and m-chlorophenylbiguanidine ( mCPBG) at the binding site of the 5-HT 3AR were investigated at an atomic level. Site-directed mutagenesis studies in Loop B and E along with our earlier published results from mutations within Loops A, C, and D provide comprehensive data on the interaction of 5-HT and mCPBG with 5-HT 3ARs. Using this data we have constructed a refined homology model of the 5-HT 3AR that considers all of the available experimental data. 5-HT and mCPBG were docked into the newly constructed homology model and the amino acid residues critical in binding of these agonists were compared and analyzed. Our docking results reveal many similar binding interactions for 5-HT and mCPBG. Namely, residues THR181, TRP183, PHE226, ILE228, TYR234 and GLU129 were all found to play key roles in binding of both 5-HT and mCPBG. However, the results also revealed two important differences that exist between the interactions of the two agonists. In our model, a hydrogen bond is formed between the indole hydrogen of 5-HT and the residue TYR153. This interaction is not present in the case of mCPBG. Conversely, a hydrogen bond exists between SER182 and a protonated nitrogen of mCPBG, which does not exist in 5-HT. Our modeling results were found to be in accordance with experimental data.

On the Role of the First Transmembrane Domain in Cation Permeability and Flux of the ATP-gated P2X2 Receptor

P2X receptors are a family of seven ligand-gated ion channels (P2X1-P2X7) that open in the presence of ATP. We used alanine-scanning mutagenesis and patch clamp photometry to study the role of the first transmembrane domain of the rat P2X2 receptor in cation permeability and flux. Three alanine-substituted mutants did not respond to ATP, and 19 of the 22 functional receptors resembled the wild-type receptor with regard to the fraction of the total ATP-gated current carried by calcium or the permeability of calcium relative to cesium. The remaining three mutants showed modest changes in calcium dynamics. Two of these occurred at sites (Gly30 and Phe44) that are unlikely to interact with permeating cations in a meaningful way. The third was a conserved tyrosine (Tyr43) that may form an inter-pore binding site for calcium. The data suggest that, with the possible exception of Tyr43, the first transmembrane domain contributes little to the permeation properties of the P2X2 receptor.

Modeling and Systemization of Cultured Cell for High Through-Put Analysis (HTA)

In order to have practical tactics on neural drug high through-put analysis (HTA), the authors have created a new bioassay method for determining the functions of neural ion-gated channel receptors. The synapse model cell is prepared a novel cellular biosensing concept. Cloned ligand-gated ion channel receptors are integrated into insect cells, because they are easier to cultivate than mammalian cells. The engineered insect cell can express ligand-gated glutamate receptor (iGluR) on its cell surface as similar as on a post-synapse membrane. The ligand-gated ion channel receptors on the insect cell can perform functions similar to those performed on the native neural synapse membrane. We have used outer cell potential measurement to record agonist-dependent cellular responses in model cell to express ligand-gated ion channel receptor on microelectrode surface. The iGluR lets Na+ ions transferred into the cell, and the amount of positively charged ions on the electrode surface where the cell is attached decreases transiently. The temporary scarcity of positively charged ions causes a potential shift between the recording electrode and the reference electrode. Because the potential profile reflects the channel gate function very well, it provides information on the efficacy of transmitter analogs, information which can then be used for designing or assessing the efficacy of clinical drugs. This synapse model cell system is a good example of the development of advanced cellular biosensing techniques.

Ivermectin resistance and overview of the Consortium for AnthelminticResistance SNPs

Ivermectin (IVM) has transformed nematode parasite control in veterinary medicineand the control of some nematode infections in humans, such as onchocerciasais,lymphatic filariasis in Africa and strongyloidiasis. Unfortunately, IVMresistance is now a serious problem for parasite control in livestock and thereis a concern about resistance development and spread in nematode parasites ofhumans. IVM is believed to act by opening glutamate-gated chloride channels andGABA-gated channels in invertebrate neurons or muscle cells, leading tohyperpolarisation of the cells and to an inhibitory paralysis. However, in thefilarial nematodes, it is not altogether clear that the effect of IVM isconfined to these actions or even whether these are the most important.Alterations in some ligand-gated ion channel (LGIC) receptor subunits may play arole in the mechanisms of IVM resistance in some nematodes, but the evidencethat changes in LGICs are the most important cause of IVM resistance innematodes is far from clear. What is evident is that IVM is an excellentsubstrate for some ATP-binding cassette transporters, IVM selects for changes inexpression levels of ABC transporters, such as P-glycoproteins, and that alteredlevels of some ABC transporters contribute to IVM resistance. In addition, thereis growing evidence that IVM selects on β-tubulin, at least in somenematodes. Based on these various mechanisms, which contribute to IVMresistance, it may become possible to develop panels of molecular markers forIVM resistance in different nematode parasites. In order to stimulate thedevelopment of such markers, an international Consortium for AnthelminticResistance SNPs (CARS) has been developed to help coordinate marker development,advance our knowledge of helminth biology and possibly assist with thedevelopment of new anthelmintic molecules.

Molecular Pharmacology of the Glycine Receptor Chloride Channel

The glycine receptor (GlyR) Cl(-) channel belongs to the cysteine-loop family of ligand-gated ion channel receptors. It is best known for mediating inhibitory neurotransmission in motor and sensory reflex circuits of the spinal cord, although glycinergic synapses are also present in the brain stem, cerebellum and retina. Extrasynaptic GlyRs are widely distributed throughout the central nervous system and they are also found in sperm and macrophages. A total of 5 GlyR subunits (alpha1-4 and beta) have been identified. Embryonic receptors comprise alpha2 homomers whereas adult receptors comprise predominantly alpha beta heteromers in a 2:3 stoichiometry. Notably, the alpha3 subunit is present in synaptic GlyRs that mediate inhibitory neurotransmission onto spinal nociceptive neurons. These receptors are specifically inhibited by inflammatory mediators, implying a role for alpha3-containing GlyRs in inflammatory pain sensitisation. Because molecules that increase GlyR current may have clinical potential as muscle relaxant and peripheral analgesic drugs, this review focuses on the molecular pharmacology of GlyR potentiating substances. Of all GlyR potentiating substances identified to date, we conclude that 5HT(3)R antagonists such as tropisetron offer the most promise as therapeutic lead compounds. However, one problem is that that virtually all known GlyR potentiating compounds, including tropisetron analogues, lack specificity for the GlyR. Another is that almost nothing is known about the pharmacological properties of alpha3-containing GlyRs, which is the subtype of choice for targeting by novel antinociceptive agents. These issues need to be addressed before GlyR-specific therapeutics can be developed.

Effects of CDP-choline and the combination of CDP-choline and galantamine differ in an animal model of schizophrenia: Development of a selective α 7 nicotinic acetylcholine receptor agonist strategy

The regionally selective reduction of expression of the α 7 nicotinic acetylcholine receptor (α 7 nAChR) in schizophrenia underlies impaired sensory inhibition, a possible endophenotype of the disorder. This ligand-gated ion channel receptor has been proposed as a pharmacotherapeutic target in schizophrenia. The current study examined the effect of CDP-choline alone and the combination of CDP-choline and galantamine, administered acutely and once-daily for five consecutive days, in an animal model of NMDA receptor hypofunction that is relevant to schizophrenia. The results support the allosteric modulatory influence of galantamine on CDP-choline; however, individual doses of CDP-choline and galantamine must be carefully titrated in order to achieve optimal levels of α 7 nAChR “agonism” that may be necessary for the desired therapeutic effect.

Purine and pyrimidine receptors.

Adenosine 5'-triphosphate (ATP), in addition to its intracellular roles, acts as an extracellular signalling molecule via a rich array of receptors, which have been cloned and characterised. P1 receptors are selective for adenosine, a breakdown product of ATP, produced after degradation by ectonucleotidases. Four subtypes have been identified, A(1), A(2A), A(2B) and A(3) receptors. P2 receptors are activated by purines and some subtypes also by pyrimidines. P2X receptors are ligand-gated ion channel receptors and seven subunits have been identified, which form both homomultimers and heteromultimers. P2Y receptors are G protein-coupled receptors, and eight subtypes have been cloned and characterised to date.

Assembly of ligand-gated ion channel receptors – how individual subunits contribute to receptor function

Assembly of ligand-gated ion channel receptors – how individual subunits contribute to receptor function

Ligand-Induced 5-HT3 Receptor Internalization in Enteric Neurons in Rat Ileum

Release of 5-hydroxytryptamine (5-HT) from mucosal enterochromaffin cells and activation of 5-HT(3) receptors (5-HT(3)Rs) on neurons in the gut wall is important in the response of the gut to the luminal environment. Intestinal inflammation is associated with increased levels of mucosal 5-HT. The aims of the study were to determine the following: (1) if 5-HT(3)R undergoes ligand-induced internalization in myenteric neurons, and (2) the effect of long-term increase of mucosal 5-HT on 5-HT(3)Rs. Acute effects of exogenous 5-HT or endogenous release of 5-HT by luminal glucose on cellular localization of 5-HT(3)Rs was determined by immunohistochemistry and confocal microscopy. Treatment with the serotonin re-uptake inhibitor, fluoxetine, for 6 days (20 mg/kg daily orally) was used to increase mucosal 5-HT chronically in rats. Net ileal fluid movement was measured in anesthetized rats by the weight change of a 2.5% agarose cylinder. Acute increases in 5-HT induced by exogenous or endogenous 5-HT decreased 5-HT(3)R immunoreactivity at the neuronal cell membrane by 70% and 60%, respectively. Chronic fluoxetine treatment increased mucosal levels of 5-HT and decreased membrane 5-HT(3)R immunoreactivity by 27%. Net fluid absorption was decreased by a 5-HT(3)R agonist or by luminal glucose; this was attenuated 88% and 99%, respectively, by fluoxetine treatment. Long-term increase in 5-HT in the intestinal mucosa results in increased 5-HT(3)R internalization in myenteric neurons. Chronic changes in mucosal 5-HT may alter gastrointestinal secretory and motor function via ongoing loss of receptor from neuronal membrane, causing a mismatch between luminal content and absorption.

In Vitro Antagonism of Recombinant Ligand-Gated Ion-Channel Receptors by Stereospecific Enantiomers of Bupivacaine

Bupivacaine is a racemic mixture of S(-)- and R(+)-enantiomers. Both isomers have similar potency as local anesthetics, but the S(-)-enantiomer produces less central nervous system and cardiovascular toxicity. Local anesthetic-induced convulsion is likely to be associated with not only sodium channel but also ligand-gated ion channel. The present study investigates the direct effects of the stereoenantiomers of bupivacaine on 4 recombinant ligand-gated ion-channel receptors. The antagonist activities of the S(-)- and R(+)-enantiomers of bupivacaine were tested at the nicotinic acetylcholine, N-methyl-d-aspartate (NMDA), gamma-aminobutyric acid(A) (GABA(A)), and 5-hydroxytryptamine(3A) (5-HT(3A)) receptors expressed in Xenopus oocytes using a 2-voltage clamp technique. Racemic bupivacaine and its 2 enantiomers all antagonized the 4 receptors in a concentration-dependent manner. Potencies at nicotinic acetylcholine, NMDA, and 5-HT(3A) receptors were similar. At GABA(A) receptors, the potency of R(+)-bupivacaine was less than racemic bupivacaine or levobupivacaine. Comparison of the antagonist potencies with local concentrations obtained in clinical use suggests that bupivacaine and its enantiomers are likely to produce extensive inhibition at the nicotinic acetylcholine, NMDA, and 5-HT(3A) receptors but a much weaker and probably not clinically relevant effect at the GABA(A) receptor. It is possible that direct effects at these receptors may contribute, at least in part, to the spinal and epidural anesthesia induced by these compounds. It is unlikely, however, that the difference of the toxicity in bupivacaine enantiomers is because of the stereoselectivities of bupivacaine at ligand-gated ion-channel receptors studied.

In Vitro Antagonism of Recombinant Ligand-Gated Ion-Channel Receptors by Stereospecific Enantiomers of Bupivacaine

In Vitro Antagonism of Recombinant Ligand-Gated Ion-Channel Receptors by Stereospecific Enantiomers of Bupivacaine