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The Structural Basis for Ligand Recognition by Mouse Odorant ReceptorsRepicky, Sarah Elizabeth 22 April 2008 (has links)
Mammalian odorant receptors (ORs) are Class I G-protein coupled receptors (GPCRs) located within the nasal epithelium. Odorant receptors interact with Galpha olfactory, a Galpha S type G-protein. Activated Galpha olfactory stimulates adenylate cyclase and the resulting increase in cAMP concentration opens cyclic nucleotide gated channels allowing Ca2+ to enter the cell. The increased Ca2+ then activates a Ca2+ activated Cl- channel which further depolarizes the cell. This depolarization initiates an action potential that reaches the axon of the olfactory sensory neuron located in the main olfactory bulb. Information from the main olfactory bulb is then transmitted to higher regions of the brain. Olfactory information is initially coded through the interaction of odorant molecules with hundreds of distinct ORs, but difficulty in exogenous expression of odorant receptors has delayed the identification of ligands for individual ORs. However, expression of mouse odorant receptors in Xenopus laevis oocytes allows for a systematic screening for potential ligands, as well as for efficient study of the structure-function relationship of the receptors and their ligands. My screening of odorant receptors using Xenopus oocytes included the coexpression of a signal transduction system and the use of robotic two-electrode voltage clamp electrophysiology. In this study, I investigated the structural basis for ligand recognition in mouse odorant receptors. First, I expanded the molecular receptor ranges of seven Class I odorant receptors. By use of a high throughput assay, I was able to expand upon current knowledge in the field for the mouse odorant receptors 23-1, 31-4, 32-11, 40-4, 42-1, 42-2 and 42-3. I then examined one receptor (MOR23-1) in more detail. I used the substituted cysteine accessibility method to identify residues within transmembrane domain five of this receptor that are accessible from the extracellular space. These residues may line the ligand binding site or the ligand access pathway. Conventional mutations of A205 caused little alteration in the molecular receptive range of the receptor, suggesting that this residue may not play a significant role in ligand interaction within the binding pocket. Mutagenesis of G111, a residue within transmembrane domain three caused significant shifts in the molecular receptive range of the receptor, but the location of this residue within the binding pocket could not be confirmed by the substituted cysteine method. Previous reports had suggested significant similarity between the molecular receptive ranges of the seven mouse odorant receptors that I used in my research. By expanding upon the known aliphatic ligands for each receptor identified new ligands for each receptor, I was able to show that the molecular receptive ranges of these receptors are in fact distinct. The experimental identification of residues located within the binding pocket on transmembrane five of mouse odorant receptor 23-1 provides an improved understanding of ligand recognition by this receptor class and will aid in better computer modeling of these receptors. This increased accuracy of the computer models of these basic Class I GPCRs may aid in future drug discoveries. Since GPCRs constitute a significant fraction of current drug targets, understanding the mechanism of ligand interactions with mouse odorant receptors may aid in the development of more efficacious compounds in the treatment of many common ailments.
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Nicotinic acetylcholine receptors from the parasitic nematode Ascaris suumWilliamson, Sally January 2008 (has links)
Nematodes of the genus Ascaris are large gastrointestinal parasites. Ascaris lumbricoides infects ~1 billion people globally; causing malnutrition and general morbidity, and can block the gut or bile duct causing fatal complications. Ascaris suum is a parasite of pigs; in addition to its veterinary significance, it can occasionally be zoonotic, and is a good model of the human parasite. One of the main classes of drugs used to treat parasitic nematode infections are the cholinergic anthelmintics, such as levamisole and pyrantel, which act as agonists of nicotinic acetylcholine receptors at the nematode neuromuscular junction.
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A Study of Electrogenic Transient and Steady-state Cotransporter Kinetics: Investigations with the Na+/Glucose Transporter SGLT1Krofchick, Daniel 31 August 2012 (has links)
Significant advancements in the field of membrane protein crystallography have provided in recent years invaluable images of transporter structures. These structures, however, are static and require complementary kinetic insight to understand how their mechanisms work. Electrophysiological studies of transporters permit the high quality kinetic measurements desired, but there are significant difficulties involved in analyzing and interpreting the data. Current methods allow a variety of kinetic parameters to be measured but there is a disconnect between these parameters and a fundamental understanding of the carrier. The intent of this research was to contribute new tools for studying the electrogenic kinetics of membrane transport proteins, to understand the link between these kinetics and the carrier, and to ultimately understand the mechanisms involved in transport. In this vein, two projects are explored covering two important kinetic time domains, transient and steady-state. The transient project studies the conformational changes of the unloaded carrier of SGLT1 through a multi-exponential analysis of the transient currents. Crystal structures have potentially identified a gated rocker-switch mechanism and the transient kinetics are used to support and study this kinetically. A protocol taking advantage of multiple holding potentials is used to measure the decay time constants and charge movements for voltage jumps from both hyperpolarizing and depolarizing directions. These directional measurements provide insight into the arrangement of the observed transitions through directional inequalities in charge movement, by considering the potential for a slow transition to hide a faster one. Ultimately, four carrier decays are observed that align with the gated rocker-switch mechanism and can be associated one-to-one with the movement of a gate and pore on each side of the membrane. The steady-state project considers a general theoretical model of transporter cycling. Recursive patterns are identified in the steady-state velocity equation that lead to a broad understanding of its geometric properties as a function of voltage and substrate concentration. This results in a simple phenomenological method for characterizing the I–V curves and for measuring the kinetics of rate limiting patterns in the loop, which we find are the basic structures revealed by the steady-state velocity.
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A Study of Electrogenic Transient and Steady-state Cotransporter Kinetics: Investigations with the Na+/Glucose Transporter SGLT1Krofchick, Daniel 31 August 2012 (has links)
Significant advancements in the field of membrane protein crystallography have provided in recent years invaluable images of transporter structures. These structures, however, are static and require complementary kinetic insight to understand how their mechanisms work. Electrophysiological studies of transporters permit the high quality kinetic measurements desired, but there are significant difficulties involved in analyzing and interpreting the data. Current methods allow a variety of kinetic parameters to be measured but there is a disconnect between these parameters and a fundamental understanding of the carrier. The intent of this research was to contribute new tools for studying the electrogenic kinetics of membrane transport proteins, to understand the link between these kinetics and the carrier, and to ultimately understand the mechanisms involved in transport. In this vein, two projects are explored covering two important kinetic time domains, transient and steady-state. The transient project studies the conformational changes of the unloaded carrier of SGLT1 through a multi-exponential analysis of the transient currents. Crystal structures have potentially identified a gated rocker-switch mechanism and the transient kinetics are used to support and study this kinetically. A protocol taking advantage of multiple holding potentials is used to measure the decay time constants and charge movements for voltage jumps from both hyperpolarizing and depolarizing directions. These directional measurements provide insight into the arrangement of the observed transitions through directional inequalities in charge movement, by considering the potential for a slow transition to hide a faster one. Ultimately, four carrier decays are observed that align with the gated rocker-switch mechanism and can be associated one-to-one with the movement of a gate and pore on each side of the membrane. The steady-state project considers a general theoretical model of transporter cycling. Recursive patterns are identified in the steady-state velocity equation that lead to a broad understanding of its geometric properties as a function of voltage and substrate concentration. This results in a simple phenomenological method for characterizing the I–V curves and for measuring the kinetics of rate limiting patterns in the loop, which we find are the basic structures revealed by the steady-state velocity.
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The Role of the M4 α-Helix in Lipid Sensing by a Pentameric Ligand-Gated Ion ChannelHénault, Camille 11 August 2021 (has links)
Pentameric ligand-gated ion channels (pLGICs) are membrane-embedded receptors found extensively in pre- and post-synaptic membranes throughout the nervous system where they play an important role in neurotransmission. The function of the prototypic pLGIC, the nicotinic acetylcholine receptor (nAChR) is highly sensitive to changes in its lipid environment, while other pLGICs display varying lipid sensitivities. This thesis presents a multidisciplinary investigation into the features of the transmembrane domain (TMD) that determine the unique functional and physical traits of different pLGICs. Using two prokaryotic homologues of the nAChR, ELIC and GLIC, as models, I focus on the outermost, lipid-exposed α-helix, M4, which, despite being distant from the primary allosteric pathway coupling agonist binding to channel gating, exercises significant control over channel function. Here, I present evidence that M4 acts as a lipid sensor, detecting changes in the surrounding lipids and transmitting these changes to the channel pore via contacts with the adjacent TMD α-helices, M1 and M3, and/or with structures in the extracellular domain. Using ELIC and GLIC chimeras, I first show that the TMD is the main driver of pLGIC thermal stability. I then demonstrate that the M4 α-helices in each channel play different roles in channel maturation and function, which suggests a divergent evolutionary path. Following this, I show that the M4 C-terminus is essential to both maturation and function in GLIC, while in ELIC its role is less defined, again showcasing possible evolutionary differences. Building on these findings, I examined the role of aromatic residues at the M4 – M1/M3 interface, and found that they predictably determine the interactions between M4 and M1/M3. Notably, the addition of aromatic residues to enhance M4-M1/M3 interactions in ELIC promotes channel function, while the elimination of aromatic residues at the M4-M1/M3 interface in GLIC is detrimental to channel function. Furthermore, I show that these same aromatics alter the strength of pLGIC lipid sensing and the sensitivity to certain disease-causing mutations, both indicating that aromatic residues are key players in channel function, stability and modulation. Finally, I and my collaborators identified and characterized a novel desensitization-linked lipid binding site in ELIC. Extensive mutagenesis studies coupled with biophysical measurements allowed us to develop a model describing how lipid binding influences the rates of ELIC desensitization to shape the agonist-induced response.
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Governing Dynamics of Divalent Copper Binding by Influenza A Matrix Protein 2 His37 ImidazoleMcGuire, Kelly Lewis 04 August 2020 (has links)
Influenza A is involved in hundreds of thousands of deaths globally every year resulting from viral infection-related complications. Previous efforts to subdue the virus by preventing proper function of wild-type (WT) neuraminidase (N), and M2 proteins using oseltamivir and amantadine (AMT) or rimantadine (RMT), respectively, exhibited success initially. Over time, these drugs began exhibiting mixed success as the virus developed drug resistance. M2 is a proton channel responsible for the acidification of the viral interior which facilitates release of the viral RNA into the host. M2 has a His37-tetrad that is the selective filter for protons. This protein has been demonstrated to be a feasible target for organic compounds. However, due to a mutation from serine to asparagine at residue 31 of M2, which is found in the majority of influenza strains circulating in humans, AMT and RMT block is insufficient. From simulations, it is unclear whether the insensitivity results from weak binding or incomplete block. The question of how the S31N mutation caused MT and RMT insensitivity in M2 is addressed here by analyzing the binding kinetics of AMT and RMT using the two-electrode voltage clamp electrophysiology method. The dissociation rate constant (k2) is dramatically increased compared to WT for both AMT and RMT, by 1500-fold and 17000-fold respectively. Testing of AMT at 10 mM demonstrates complete block, albeit weak, of the S31N M2 channel. At 10 mM, RMT does not reach complete block even though the binding site is saturated. When RMT is in the bound state, it is not blocking all the current, and is binding without block. These results motivated the development of novel M2 blockers using copper complexes focusing on the His37 complex in M2. I hypothesized that copper complexes would bind with the imidazole of a histidine in the His37 complex and prevent proton conductance. The His37 complex is highly conserved in the M2 channel and, therefore, would be important target for influenza therapeutics. By derivatizing the amines of known M2 blockers, AMT and cyclooctyalmine, to form the iminodiacetate or iminodiacetamide, we have synthesized Cu(II) containing complexes and characterized them by NMR, IR, MS, UV–vis, and inductively coupled plasma mass spectroscopy (ICP-MS). The copper complexes, but not the copper-free ligands, demonstrated H37-specific blocking of M2 channel currents and low micromolar anti-viral efficacies in both Amt-sensitive and Amt-resistant IAV strains with, for the best case, nearly 10-fold less cytotoxicity than CuCl2. Isothermal titration calorimetry was used to obtain enthalpies that showed the copper complexes bind to one imidazole and curve fitting to the electrophysiology data provided rate constants for binding in the M2 channel. Computational chemistry was used to obtain binding geometries and energies of the copper complexes to the His37-tetrad. The results show that the copper complexes do bind with the His37 complex and prevent proton conductance and influenza infection.
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Characterization of diazepam binding inhibitor as a structure-function tool for human ɣ-aminobutyric acid-A receptorsSimon-Guth, Szabolcs January 2023 (has links)
Gammaaminosmörsyrareceptorer typ A (GABAAR) är pentameriska ligandstyrda kloridkanaler som uppvisar neurohämmande egenskaper. Därmed är de primära läkemedelsmål för flera ångestdämpande och lugnande läkemedel som används för att minska förekomsten av aktionspotential i neuroner. Trots vikten av dessa receptorer har strukturen av öppen receptor för GABAAR inte lösts hittills, på grund av deras snabba desensibiliseringskinetik. Diazepambindande hämmare (DBI) är en neuropeptid som tidigare rapporterats vara en positiv modulerare för α5β3 GABAAR. I denna studie framställdes DBI genom rekombinant proteinexpression, och den positiva moduleringen undersöktes och karakteriserades med hjälp av voltage-clamp med två elektroder på Xenopus laevis oocyter. För att kunna studera DBI moduleringen skapades GABA dos-responskurvan, och dess karakteristik undersöktes. Baserat på resultaten verkar den positiva moduleringen av DBI vara koncentrationsberoende. Vidare orsakar moduleringen en 2,16-faldig ökning av GABA-framkallad ström vid dess maximala modulationskoncentration. Trots att ström signaler från voltage-clamp uppvisar en viss grad av variabilitet stämmer resultaten överens med tidigare rapporterade observationer som utredde DBI moduleringen respektive GABA dos-responskurvan för α5β3 GABAAR. Dessa resultat kan utnyttjas för att stödja framtida strukturella studier av GABAAR genom att använda denna kunskap om DBI för att potentiellt kunna stabilisera den öppna receptorn, såväl som för att förstå mekanismen för interaktionen mellan DBI och GABAAR. / γ-Aminobutyric acid type-A receptors (GABAARs) are pentameric ligand-gated chloride channels which exhibit neuro inhibitory effects. Hence, they are the primary drug-targets of multiple anxiolytic and sedative drugs used to inhibit the firing rate of neurons. Despite the importance of these receptors, the open structure of GABAAR has not been resolved, owing to their rapid desensitization kinetics. Diazepam binding inhibitor (DBI) is a neuropeptide previously reported to positively modulate the α5β3 GABAARs. In this study, DBI was recombinantly expressed, and this positive modulation was further investigated and characterized by using two-electrode voltage clamp of Xenopus oocytes. For the purpose of studying DBI modulation, GABA dose-response curve was generated, and its characteristics were assessed. Based on the results, the positive modulation of DBI appears to be concentration dependent. Furthermore, the modulation causes a 2.16-fold increase in GABA-elicited current at its maximum modulatory concentration. Although the current traces present some degree of variability, the results are supported by being consistent with previously reported findings investigating DBI modulation and the dose-response curve for α5β3 GABAARs, respectively. These findings can be used to support future structural studies of GABAARs by utilizing this knowledge of DBI to potentially stabilize the open structure of the receptor, as well as in understanding the mechanism of interaction between DBI and GABAARs.
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Einflüsse der Serum- und Glukokortikoidkinasen 1 und 3 auf den humanen Na⁺- Dikarboxylat- Transporter NaDC3 / Differential effect of the serum and glucocorticoid kinases 1 and 3 on the sodium-dependent dicarboxylate cotransporter NaDC3Dzidowski, Andrea 22 August 2017 (has links)
No description available.
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