No description available.

Recent evidence has indicated that NSAIDs might have direct effects on CNS tissue in addition to their classical inhibitory action on COX enzymes. This thesis addresses this hypothesis using electrophysiological and behavioural techniques. The effects of fenamate and other NSAIDs on native neuronal GABA(_A), 5-HT(_3), nicotinic ACh, P2x and strychinine-sensitive glycine receptors, expressed on isolated vagus or optic nerves, was investigated using an extra-cellular recording technique. The fenamate NSAID, mefenamic acid (MFA) potentiated GABA (10µM)- evoked responses in the vagus nerve. Application of MFA also resulted in non-competitive inhibition of 5-HT-and a,βMeATP- evoked responses. Non-competitive like inhibition was also observed with flufenamic acid on DMPP- and a,βMeATP- evoked responses and with meclofenamic acid on GABA- evoked responses. Non-fenamate NSAIDs, including aspirin, did not significantly modulate the GABA(_A), 5-HT(_3), nicotinic ACh, P2x or glycine receptors. The cognitive and behavioural effects of fenamates and other NASIDs were then investigated. MFA (5-20mg/kg) caused a significant dose- and time-dependent enhancement in the non-spatial object discrimination working memory task when compared to saline controls. The enhancement observed with MFA was greater than that of the cognitive enhancer piracetam. This enhancement was not due to a change in non-mnemonic processes such as arousal, anxiety or locomotion. MFA also enhanced rats' performance in the spatial object location working memory task. The fenamate NSAID, meclofenamic acid (20mg/kg) mimicked the effect of MFA, but the non-fenamate NSAIDs aspirin and ibuprofen, did not enhance object discrimination indicating that these cognitive effects are not via inhibition of COX. The GABA(_A) receptor modulators diazepam, bicuculline and loreclezole, did not replicate the effect of MFA on object discrimination, suggesting that its effects do not depend entirely on the GABAa receptor. Scopolamine (0.25-lmg/kg) significantly impaired object discrimination in a dose-dependent manner. This action could be fully reversed by co-treatment with MFA (20mg/kg).In the T-maze task, MFA (20mg/kg) decreased the number of arm entry errors and days taken to reach criterion. The number of arm entry errors made when a 5-minute intra-trial interval was introduced was also significantly reduced by MFA compared with saline treated animals. In the radial maze, MFA (20mg/kg) did not decrease the number of never baited arm entries to reach criterion. However MFA did significantly reduce the number of re-entry errors to baited arms, compared to controls, when an intra-trial delay (10-30 sees) was introduced. These results support the hypothesis that MFA enhances spatial working memory and that these effects are not task-specific. Overall, the data in this thesis show that fenamate NSAIDs can directly modulate native neuronal ligand-gated ion channels and that MFA can enhance working memory in normal and scopolamine-impaired rats. These results suggest additional pharmacological potential for certain fenamate NSAIDs.

Neuronal nicotinic acetylcholine receptors can have their function modulated by zinc. Depending on concentration and subunit composition, zinc either inhibits or potentiates receptor function. The zinc ion potentiates the alpha4beta4 receptor at non-agonist binding interfaces or "pseudo sites." Zinc potentiation is reduced if certain residues are mutated or spatially interfered with. The residue contributing most to this potentiation reduction effect is histidine 162 on the alpha4 subunit. The anthelmintic compound levamisole potentiates acetylcholine response of certain neuronal nicotinic receptors. Levamisole and its functional analogues morantel, oxantel, and pyrantel all were found to potentiate alpha4beta4 receptors at low (µM) concentrations and inhibit them at high (mM) concentrations. Oxantel showed the greatest degree of potentiation, about a third of the maximal zinc potentiation measured. Oxantel was screened using the substituted cysteine accessibility method (SCAM) against the residue histidine 162 as well as nearby alpha4 residues histidine 61 and glutamate 59 and the beta4 residue aspartate 195. Screening was carried out by mutating said residues into cysteine, followed by covalent linkage with a disulfide bridge of that residue with a methanethiosulfonate compound. SCAM experiments allowed testing of the effects of a single residue and the area immediately adjacent to it. Receptors that lost zinc potentiation capacity from site-directed mutagenesis at the his 162 residue and subsequent methanethiosulfonate reaction still showed regular potentiation following oxantel treatment. Although these compounds exhibit similar biphasic potentiation dose-response curves as zinc, their mechanism for potentiation is not through the same mechanism.

Τα πενταμερή ιοντικά κανάλια που ενεργοποιούνται από τη δέσμευση ενός προσδέτη (pLGICs-pentameric Ligand Gated Ion Channels) της υπεροικογένειας των υποδοχέων Cys-θηλιάς είναι διαμεμβρανικές γλυκοπρωτεΐνες που εμπλέκονται σε ποικιλία βιολογικών λειτουργιών. Στην παρούσα εργασία παρουσιάζεται η NMR μελέτη της εξωκυττάριας περιοχής ενός προκαρυωτικού pLGIC, της GLIC (Gloeobacter ligand-gated ion channel), που προέρχεται από το κυανοβακτήριο Gloeobacter violaceus και εμφανίζεται ως μονομερές σε διάλυμα. / Pentameric ligand-gated ion channels (pLGICs) of the Cys loop family are transmembrane glycoproteins implicated in a variety of biological functions. Here, we present a solution NMR study of the extracellular domain of a prokaryotic pLGIC homologue from the bacterium Gloeobacter violaceus that is found to be a monomer in solution.

In lipid membranes lacking activating lipids, the nicotinic acetylcholine receptor adopts an uncoupled conformation that binds ligand, but does not transition into an open conformation. Understanding the mechanisms of lipid-dependent uncoupling is essential to understanding lipid-nAChR interactions, which may be implicated in pathological conditions such as nicotine addition. Here, I tested two structural features of a proposed uncoupling method to elucidate the mechanism of lipid-dependent uncoupling. First, infrared measurements and electrophysiological characterization performed in prokaryotic homologues indicate that lipid sensitivity is largely controlled by the most peripheral α-helix in the transmembrane domain, M4. My data show that tighter association of M4 with the adjacent M1 and M3 transmembrane α-helices decreases a receptor’s propensity to adopt a lipid-dependent uncoupled conformation. Second, I indirectly tested the hypothesis that uncoupling results from a conformational change at the extracellular/transmembrane domain interface that leads to an increased separation between the two domains and ultimately to a constriction of the channel pore. Finally, biophysical studies presented in this dissertation shed light on the complex binding of a number of non-competitive channel blockers to the nicotinic acetylcholine receptor channel pore in both the resting and desensitized states. The data provide further insight into the structural rearrangements that occur upon uncoupling of ligand binding and gating in the nicotinic acetylcholine receptor.

Pentameric ligand-gated ion channels are critical mediators of electrochemical signal transduction in neurons and other excitable cells, causing them to be important targets of psychoactive drugs. Structural data for these complex proteins are limited, particularly among eukaryotic family members and for the functionally critical open state. These data limitations cause knowledge gaps regarding the mechanisms of ion channel opening, gating, and modulation. However, a newly discovered bacterial family member, known as sTeLIC, shares numerous structural features with its eukaryotic relatives in our central nervous system. A recently solved electron microscopy structure depicts sTeLIC in an apparent open state with binding pockets in its extracellular domain, compatible with binding a drug with structural similarities to amphetamines, like the 4-bromoamphetamine. This project aims to provide the first structure-function evidence for direct modulation of a pentameric ligand-gated ion channel by an amphetamine. The two most essential tools used in this project to examine the effects of 4-bromoamphetamine on sTeLIC were Xenopus laevis oocytes and two-electrode voltage-clamp. These tools were necessary for the collection of gating and modulation data. Ion channel activities can be analysed by clamping sTeLIC injected Xenopus laevis oocytes into the two-electrode voltage-clamp since it can artificially control the membrane voltage of oocytes. Modulation data show that 4-bromoamphetamine is a bimodal allosteric potentiator, as well as an allosteric agonist. Residues Y104 and W75, located in the binding pocket, were selected by comparing the published open state model with an AlphaFold-generated non-conducting model. Mutating these into valine or alanine reduces the potentiation. One explanation may be that removing tyrosine's aromatic ring complicates retaining essential interactions in the binding pocket while swapping tryptophan for smaller residues could make it easier for the drug to stabilise the closed state.

No description available.

No description available.

This thesis focuses on the structural and functional characterization of two integral membrane proteins; CfrA, an outer membrane TonB-dependent transporter (TBDT) from Campylobacter jejuni, and ELIC, a pentameric ligand-gated ion channel (pLGIC) from Erwinia Chrysanthemi. The spectroscopic characterization of CfrA revealed a fold consistent with the structural and biophysical properties observed for other TBDT. Both a homology model of CfrA and sequence alignments of CfrA with other ferric-enterobactin transporters suggested a unique mode of ligand binding, thus raising the possibility that C. jejuni can be specifically inhibited. To investigate the molecular determinates of binding to CfrA, I set out to crystallize CfrA. Hundreds of crystal trials led to crystals diffracting to 3.6 Å resolution, with a complete data set acquired at 5 Å resolution that led to a structural model of the CfrA β-barrel. In the second part of this thesis, I reconstituted ELIC into model membranes in order to test the role of intramembrane aromatic interactions in ELIC gating and lipid sensing. ELIC was reconstituted into both asolectin (aso-ELIC) and 1-palmitoyl-2-oleoyl phosphatidylcholine (PC-ELIC), membranes that stabilize the homologous nicotinic acetylcholine receptor (nAChR) in functional coupled versus non-functional uncoupled conformations, respectively. In both membrane environments, ELIC exhibits a mixed α-helical and β-sheet secondary structure, with a thermal denaturation intermediate between those of the nAChR and the close prokaryotic homolog, GLIC, in similar membranes. The data suggest that although ELIC has a decreased propensity to adopt an uncoupled conformation relative to the nAChR, its ability to undergo cysteamine-induced channel gating is sensitive to its lipid environment. The decreased propensity to uncouple may reflect an increased level of aromatics at the interface between the transmembrane α-helices, M1, M3, and M4. To test this hypothesis further, the level or aromatic residues at the M1, M3, and M4 interface in both GLIC and ELIC were varied, and in both cases the levels of intramembrane aromatic interactions correlated with the efficiency of coupling binding to gating. The data provide further evidence for a role of intramembrane aromatics in channel gating and in dictating the propensity of pentameric ligand-gated ion channels to adopt an uncoupled conformation.

Pentameric ligand-gated ion channels (pLGICs) are membrane receptors that play a crucial role in every living organism. The pLGIC protein structure forms a pore through the membrane of a cell that can let specific ions pass through, upon activation by endogenous agonists. pLGICs are allosterically modulated by ligands binding at allosteric sites, that either stabilize a certain conformation or change the binding affinity of the endogenous agonist. However, much remains unknown about the exact way in which these modulators bind to and affect pLGICs. An increased understanding could help in the search for novel and/or more effective target drugs. With this masters thesis, I hope to contribute by investigating the modulatory effect of ethanol on the bacterial Gloeobacter ligand-gated ion channel (GLIC). This has been done by performing oocyte electrophysiology recordings and analysis of molecular dynamics simulations, both with and without ethanol, and of four separate variants of GLIC that are either potentiated or inhibited by ethanol. Two possible allosteric sites were discovered in a transmembraneintrasubunit pocket: a potentiating allosteric site close to the M2 helix and residue V242, as well as an inhibitory membrane- and M4 helix-close intrasubunit site. Finally, evidence was found that could support a previously suggested inhibitory allosteric site in the pore around the 9’ hydrophobic gate. / Pentameriska ligandstyrda jonkanaler (pLGICs) är membranreceptorer som utgör vitala delar av varje levande organism. pLGICs proteinstruktur formar en por genom cellmembranet, som kan släppa igenom specifika joner efter aktivering av endogena agonister. pLGICs är allostermodulerade av ligander som binder vid allostera säten och som därigenom antingen stabiliserar en viss form eller förändrar den endogena agonistens bindningsstyrka. Emellertid saknas fortfarande mycket kunskap på detaljnivå om hur dessa modulatorer binder sig till och påverkar kanalerna. En ökad förståelse skulle hjälpa forskningen efter nya och/eller mer effektiva mediciner. Mitt examensarbetehoppas bidra genom att studera hur etanol modulerar den bakteriella ligandstyrda jonkanalen GLIC från Gloeobacter. Det har gjorts genom elektrofysiologimätningar på oocyter och analys av molekulärdynamiksimuleringar, båda av fyra olika GLIC-varianter, som antingen potentieras eller hämmas av etanol, och med eller utan etanol. Två allostera säten upptäcktes i det transmembrana intrasubenhetområdet: ett säte för potentiering nära M2 helixen och aminosyran V242, och ett hämmande säte nära membranet och helix M4. Slutligen hittades tecken som kan styrka existensen av det tidigare föreslagna hämmande allostera sätet i poren kring den hydrophoba porten.