The glycine receptor (GlyR) is a ligand-gated ion channel member of the Cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Zinc is a potent allosteric modulator of GlyR function, enhancing GlyR activity at low nM to 10[mu]M concentrations while inhibiting GlyR activity at higher concentrations. We investigated sources of contaminating zinc, identifying low nM levels of zinc in ultrapure H₂O, powdered reagents used in the preparation of common electrophysiological buffers, and in polystyrene pipets. These low levels of zinc were capable of enhancing GlyR function. These findings suggest that without checking for this effect using a zinc-chelator such as tricine, one cannot assume that responses elicited by glycine applied alone are not necessarily also partially due to some level of allosteric modulation by zinc. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of zinc with drugs of abuse on taurine-activated GlyR to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by drugs of abuse, indicating no synergistic effects on efficacy when receptors are saturated with taurine as may be seen during synaptic events in vivo. Finally, we utilized phage display to identify novel peptide modulators of the GlyR. We tested 26 peptides against [alpha1beta] GlyRs, identifying peptides with various levels of activity on GlyR function. We demonstrated that these modulators were zinc-dependent, as their effects on GlyR activity were abolished in the presence of the zinc-chelating agent tricine. Together, these data indicate the importance of accounting for the effects of zinc when studying the function of the GlyR, as even low levels of zinc that can be found as contaminants in labware and buffers can affect GlyR function and responses to various allosteric modulators, including drugs of abuse. / text

The glycine receptor (GlyR) mediates fast inhibitory neurotransmission in the central nervous system (CNS). Although GlyR α1 subunits are widely distributed, α3 subunits are found only on spinal cord pain sensory neurons where they mediate central inflammatory pain sensitization. Thus, the α3 subunit is a potential therapeutic target for anti-inflammatory analgesia. It is yet to be understood why α3 subunits are represented in these synapses. Thus, α3 subunit-specific modulators are required both as therapeutic leads and as pharmacological probes for basic research. The Thesis, which consists of three independent studies, investigated the molecular pharmacology of three classes of compounds at GlyRs, especially those containing the α3 subunit. The dihydropyridines (DHPs), nifedipine and nicardipine, modulate native GlyRs at micromolar concentrations. Nicardipine has a biphasic potentiating and inhibitory effect, whereas nifedipine causes inhibition only. The first study investigated the molecular mechanism by which these compounds inhibit recombinant GlyRs. The rate of onset of inhibition in the open state was accelerated by pre-application of DHP in the closed state, with the degree of acceleration proportional to the concentration of pre-applied DHP. This implies a non-inhibitory binding site close to the DHP inhibitory site. DHP inhibition was use-dependent and independent of glycine concentration, consistent with a pore-blocking mode of action. DHP sensitivity was abolished by the G2’A mutation, providing a strong case for DHP binding site in the pore. Nifedipine exhibited an approximately 10-fold higher inhibitory potency at α1-containing relative to α3-containing receptors, whereas nicardipine was only weakly selective for α1-containing GlyRs. The differential sensitivities of nifedipine and nicardipine for different GlyR isoforms suggest that DHPs may be a useful resource to screen as pharmacological tools for selectively inhibiting different synaptic GlyR isoforms. To date there are few compounds known to pharmacologically discriminate between α1 and α3 subunit-containing GlyRs. The second study stemmed from an observation that β-alanine and taurine act as weak partial agonists of α3 GlyRs but as strong partial agonists at α1 GlyRs. Using chimeras of α1 and α3 subunits, we identified the relatively structurally divergent M4 transmembrane domain and C-terminal tail as a specific determinant of the efficacy difference. As mutation of individual non-conserved M4 residues had little influence on agonist efficacies, the reduced efficacy of α3 GlyRs is most likely a distributed effect of all non-conserved M4 residues. Given the lack of contact between M4 and other transmembrane domains, the efficacy differences are probably mediated by differential interactions between the respective M4 domains and the surrounding lipid environment. The strong influence of M4 primary structure on partial agonist efficacy suggests that the relatively poorly conserved α3 GlyR M4 domain may be a promising domain to target in the search for α3 GlyR-specific modulators. β-carbolines have recently been shown to inhibit glycine receptors in a subunit-specific manner. The third study screened four structurally similar β-carbolines, harmane (HM), tryptoline (TP), norharmane (NHM) and 6-methoxyharmalan (MH) at recombinantly expressed α1, α1β, α2 and α3 glycine receptors. The four compounds exhibited only weak subunit-specificity, rendering them unsuitable as pharmacological probes. Because they displayed competitive antagonist activity, we investigated the roles of known glycine binding residues in coordinating the four compounds. The structural similarity of the compounds, coupled with the differential effects of C-loop mutations (T204A, F207Y) on compound potency, implied direct interactions between variable β-carboline groups and mutated residues. Mutant cycle analysis employing HM and NHM revealed a strong pairwise interaction between the HM methyl group and the C-loop in the region T204 and F207. These results, which define the orientation of the bound β-carbolines, were supported by molecular docking simulations. The information may also be relevant to understanding the mechanism of β-carboline binding to GABAAR where they are potent pharmacological probes. The identification of compounds that specifically abolish α3 GlyR-mediated currents should provide a useful means to investigate the physiological roles of this subunit. Drugs that potently and selectively enhance α3-subtype GlyR function may potentially serve as lead compounds since α3-subtype GlyRs have emerged as a potential therapeutic target for pain treatment. Results from studies forming the Thesis have identified several structural elements that might be useful for developing novel α3 subunit-specific drugs in the future.

Les récepteurs canaux pentamériques (RCPs) assurent la transmission synaptique dans le système nerveux, via des réorganisations structurales allostériques globales couplant la liaison de neurotransmetteur à l'ouverture et à la désensibilisation du canal ionique. Sur le RCP bactérien modèle GLIC, j'ai suivi ces changements conformationnels à plusieurs endroits de la protéine, par incorporation de couples fluorophore/quencher (bimane/tryptophane). Les données en détergent et en liposomes, à l'équilibre et en cinétique, m'ont permis d'identifier, et de caractériser structuralement, un intermédiaire rapide (milliseconde) de pré-activation, montrant une réorganisation majeure du domaine synaptique suite à l'application d'agoniste, mais où le pore reste fermé, et ainsi de proposer un modèle global des transitions d'activation et de désensibilisation. En combinant mutagenèse, électrophysiologie et approches structurales, j'ai également identifié une région critique, à l'interface entre les domaines extracellulaires et transmembranaires, pour l'activation et la désensibilisation. Enfin, un criblage fonctionnel m'a permis d'identifier de nouveaux modulateurs allostériques de GLIC.Mon travail de thèse contribue donc à la compréhension du mécanisme allostérique de GLIC, et présente de nouveaux outils pour l'étude des récepteurs du système nerveux humain. / Pentameric ligand-gated ion channels (pLGICs) are responsible for the synaptic transmission in the nervous system, occurring through their structural reorganizations allosterically coupling neurotransmitter binding to the opening or desensitization of the ion channel.Using the bacterial pLGIC model GLIC, I followed these conformational changes in several regions of the protein, through the incorporation of fluorophore / quencher pairs (bimane / tryptophan). The acquisition of data in detergent and liposomes, at equilibrium and in real time, allowed me to identify and structurally characterize a fast (millisecond) pre-activation intermediate. This new intermediate state is characterized by a major reorganization of the synaptic domain following agonist application, but with a closed pore, thus providing a comprehensive model for activation transitions and desensitization.Combining mutagenesis, electrophysiological and structural approaches, I also identified a critical region at the interface between the extracellular and transmembrane domains for activation and desensitization. Finally, a functional screening allowed me to identify new allosteric modulators of GLIC.This work contributes to the understanding of the allosteric mechanism of GLIC, and provides a structural template as well as new tools for the study of receptors in the human nervous system.

Les canaux ioniques pentamériques activables par un ligand (pLGIC) sont l'une des principales familles de canaux transmembranaires. Ils permettent la transduction rapide du signal dans le système nerveux central et périphérique via la liaison de neurotransmetteurs. Les pLGIC sont également présents chez les archées et les bactéries. Seuls deux pLGIC bactériens ont été caractérisés biochimiquement et structurellement jusqu'à présent (GLIC et ELIC). Ils servent de modèle d’étude à de nombreux scientifiques et ont été largement étudiés aussi bien au niveau fonctionnel que structural. Dans la première partie de mon travail de thèse, j'ai purifié, cristallisé et résolu la structure cristalline d'un nouveau pLGIC originaire d'un symbiote de gamma-protéobactérie de Tevnia jerichonana (sTeLIC). Des expériences fonctionnelles montrent que sTeLIC est activé par un pH alcalin, est sélectif pour les ions cationiques monovalents et inhibé par les cations divalents. La structure cristalline résolue à pH 8,0 présente un pore largement ouvert qui est le premier de ce type à être caractérisé dans cette famille pLGIC. De plus, nous avons identifié un modulateur fortement positif qui se lie au "site vestibulaire" dans le domaine extracellulaire, et nous avons résolu la structure cristalline de ce complexe. Des expériences fonctionnelles montrent également que sTeLIC partage de nombreuses fonctionnalités avec ELIC. ELIC et sTeLIC constitutent les archétypes d’une nouvelle classe de pLGICs, dont la forme active se caractérise par un pore largement plus ouvert que les autres pLGICs.Dans la deuxième partie de mon travail de thèse, les résidus senseurs de protons dans GLIC ont été cartographiés, afin de déterminer comment la liaison du proton stabilise l'état ouvert de GLIC. Tous les résidus titrables de GLIC ont été cartographiés par mutagenèse dirigée afin de découvrir des capteurs de protons impliqués dans le processus de déclenchement. Nous avons ainsi démontré que la résidu E35 est un résidu clé, dont la forme chargée stabilise l’état de repos, et la forme protonée l'état actif. Nous avons également démontré que la réponse au proton dépend de deux réseaux distincts à l'interface ECD-TMD qui stabilisent l'état ouvert de GLIC. Dans la troisième partie, j'ai cloné, purifié, cristallisé et déterminé les structures cristallines des formes ouvertes et fermées de DeCLIC, un pLGIC de la protéobactérie Desulfofustis. Chaque sous-unité contient un grand domaine additionnel N-terminal constitué de deux sous-domaines (NTD1 et NTD2). Il s’agit de la première structure d’un pLGIC qui contient un domaine supplémentaire extracellulaire non-canonique. / Ligand-gated pentameric ion channels (pLGIC) are one of the major families of transmembrane receptors. They allow rapid signal transduction in the central and peripheral nervous systems via neurotransmitters binding. PLGICs are also present in archaea and bacteria. Only two bacterial pLGICs have been biochemically and structurally characterized so far (GLIC and ELIC). They serve as working models for many scientists and have been extensively studied both at the functional and structural levels. In the first part of my thesis, I purified, crystallized and solved the crystal structure of a new pLGIC from gamma-proteobacterial symbionts of Tevnia jerichonana (sTeLIC). Functional experiments show that sTeLIC is activated by alkaline pH, and is selective for monovalent cationic ions and inhibited by divalent cations. The crystal structure solved at pH 8.0 displays a widely open pore that is the first of this kind to be characterized in the pLGIC family. In addition, we identified a strongly positive modulator that binds to the "vestibule site" in the extracellular domain, and we solved the crystal structure of this complex. Functional experiments show that sTeLIC shares many features with ELIC. ELIC and sTeLIC are the archetypes of a new class of pLGICs, whose active form is characterized by a much more open pore than other pLGICs. In the second part of my thesis, the proton sensor residues in GLIC have been mapped. All titratable GLIC residues were tested by site-directed mutagenesis to discover proton sensors involved in the triggering process. We have demonstrated that the residue E35 is a key residue, whose charged form stabilizes the resting state, and the protonated form the active state. We have also demonstrated that the proton response is dependent on two distinct networks at the ECD-TMD interface, which stabilize the open state of GLIC.In the third part of my thesis, I cloned, purified, crystallized and determined the crystal structures of the open and closed forms of DeCLIC, a pLGIC of Desulfofustis proteobacterium. Each subunit contains a large N-terminal additional domain consisting of two subdomains (NTD1 and NTD2). This is the first structure of a pLGIC which contains a non-canonical additional extracellular domain.

Protein interactions are the basis of synaptic function, and studying these interactions on a molecular level is crucial for understanding basic brain function, as well as mechanisms underlying neurological disorders. In this thesis, kinetic and mechanistic characterization of synaptic protein interactions was performed by using surface plasmon resonance biosensor technology. Fragment library screening against the reverse transcriptase of HIV was included, as it served as an outlook for future drug discovery against ligand-gated ion channels. The protein-protein interaction studies of postsynaptic Ca2+ -binding proteins revealed caldendrin as a novel binding partner of AKAP79. Caldendrin and calmodulin bind and compete at similar binding sites but their interactions display different mechanisms and kinetics. In contrast to calmodulin, caldendrin binds to AKAP79 both in the presence and absence of Ca2+ suggesting distinct in vivo functional properties of caldendrin and calmodulin. Homo-oligomeric β3 GABAA receptors, although not yet identified in vivo, are candidates for a histamine-gated ion channel in the brain. To aid the identification of the receptor, 51 histaminergic ligands were screened and a unique pharmacology was determined. A further requirement for identifying β3 receptors in the brain, is the availability of specific high-affinity ligands. The developed biosensor assay displayed sufficient sensitivity and throughput for screening for such ligands, as well as for being employed for fragment-based drug discovery. AMPA receptors are excitatory ligand-gated ion channels, involved in synaptic plasticity, and modulated by auxiliary proteins. Previous results have indicated that Noelin1, a secreted glycoprotein, interacts with the AMPA receptor. By using biochemical methods, it was shown that Noelin1 interacts directly with the receptor. The kinetics of the interaction were estimated by biosensor analysis, thereby confirming the interaction and suggesting low nanomolar affinity. The results provide a basis for functional characterization of a novel AMPA receptor protein interaction. The results demonstrate how secrets of synaptic protein interactions and function were revealed by using a molecular based approach. Improving the understanding of such interactions is valuable for basic neuroscience. At the same time, the technical advancements that were achieved to study interactions of ligand-gated ion channels by surface plasmon resonance technology, provide an important tool for discovery of novel therapeutics against these important drug targets.

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.

GLIC är en pH-känslig pentamerisk ligandstyrd jonkanal (pLGIC) som finns i cellmembranet hos prokaryoten Gloeobacter violaceus. GLIC är en bakteriell homolog till flera receptorer som är viktiga i nervsystemet hos de flesta eukaryotiska organismer. Dessa receptorer fungerar som mallar för utvecklingen av målstyrda bedövnings- och stimulerande läkemedel som påverkar nervsystemet. Förståelsen av ett proteins mekanismer har därför hög prioritet inför läkemedelsutvecklingen. Eukaryota pLGICs är dock mycket komplexa eftersom några av de är heteromera, har flera domäner, och de pågår eftertranslationella ändringar. GLIC, å andra sidan, har en enklare struktur och det räcker att analysera strukturen av en subenhet - eftersom alla subenheter är helt lika. Flertalet möjliga grindmekanismer föreslogs av vetenskapen men riktiga öppningsmekanismen av GLIC är fortfarande oklar. Projektets mål är att genomföra maskininlärning (ML) för att upptäcka nya grindmekanismer med hjälp av datormetoder. Urspungsdatan togs från tidigare forskning där andra ML-redskap såsom molekyldynamik (MD), elastisk nätverksstyrd Brownsk dynamik (eBDIMS) och Markovstillståndsmodeller (MSM) användes. Utifrån dessa redskap simulerades proteinet som vildtyp samt med funktionsförstärkt mutation vid två olika pH värden. Fem makrotillstånd byggdes: två öppna, två stängda och ett mellanliggande. I projektet användes ett annat ML redskap: KL-divergens. Detta redskap användes för att hitta skillnader i avståndfördelning mellan öppet och stängt makrotillstånd. Utifrån ursprungsdatan byggdes en tensor som lagrade alla parvisa aminosyrornas avstånd. Varje aminosyrapar hade sin egen metadata som i sin tur användes för att frambringa alla fem avståndsfördelningar fråm MSMs som byggdes i förväg. Sedan bräknades medel-KL-divergens mellan två avståndfördelningar av intresse för att filtrera bort aminosyropar med överlappande avståndsfördelningar. För att se till att aminosyror inom aminosyrapar som låg kvar kan påverka varandra, filtrerades bort alla par vars minsta och medelavstånd var stora. De kvarvarande aminosyroparen utvärderades i förhållande till alla fem makrotillstånd Viktiga nya grindmekanismer som hittades genom både KL-divergens och makrotillståndsfördelningar innefattade loopen mellan M2-M3 helixarna av en subenhet och både loopen mellan sträckor β8 och β9 (Loop F)/N-terminal β9-sträckan och pre-M1/N-terminal M1 av närliggande subenheten. Loopen mellan sträckor β8 och β9 (Loop F) visade höga KL-värden också med loopen mellan sträckor β1 och β2 loop samt med loopen mellan sträckor β6 och β7 (Pro-loop) och avståndet mellan aminosyror minskade vid kanalens grind. Övriga intressanta grindmekanismer innefattade parning av aminosyror från loopen β4-β5 (Loop A) med aminosyror från sträckor β1 och β6 samt böjning av kanalen porangränsande helix. KL-divergens påvisades vara ett viktigt redskap för att filtrera tillgänglig data och de nya grindmekanismer kan bli användbara både för akademin, som vill reda ut GLIC:s fullständiga grindmekanismer, och läkemedelsföretag, som letar efter bindningsställen inom molekylen för att utveckla nya läkemedel. / GLIC is a transmembrane proton-gated pentameric ligand-gated ion channel (pLGIC) that is found in the prokaryote Gloeobacter violaceus. GLIC is the prokaryotic homolog to several receptors that are found in the nervous system of many eukaryotic organisms. These receptors are targets for the development of pharmaceutical drugs that interfere with the gating of these channels - such drugs involve anesthetics and stimulants. Understanding the mechanism of a drug’s target is a high priority for the development of a novel medicine. However, eukaryotic pLGICs are complex to analyse, because some of them are heteromeric, have more domains, and because of their post-translational modifications (PTMs). GLIC, on the other hand, has a simpler structure and it is enough to study the structure of only one subunit - since all subunits are identical. Several possible gating mechanisms have been proposed by the scientific community, but the complete gating of GLIC remains unclear. The goal of this project is to implement machine learning (ML) to discover novel gating mechanisms by computational approaches. The starting data was extracted from a previous research where computational tools like unbiased molecular dynamics (MD), elastic network-driven Brownian Dynamics (eBDIMS), and Markov state models (MSMs) were used. From those tools, the protein was simulated in wild-type and in a gain-of-function mutation at two different pH values. Five macrostates were constructed: two open, two closed, and an intermediate. In this project another ML tool was used: KL divergence. This tool was used to score the difference between the distance distributions of one open and one closed macrostate. The starting data was used to create a tensor that stored all residue-residue distances. Each residue pair had its own metadata, which in turn was used to yield the distance distributions of all five pre-build MSMs. Then the average KL scores between two states of interest were calculated and were used to filter out the residue pairs with overlapping distance distributions. To make sure that the residues within a pair can interact with each other, all residue pairs with very high minimum and average distance were filtered out as well. The residue pairs that remained were later evaluated across all five macrostates for further studies. Important novel mechanisms discovered in this project through both the KL divergence and the macrostate distributions involved the M2-M3 loop of one subunit and both the β8-β9 loop/N-terminal β9 strand and the preM1/N-terminal M1 region of the neighboring subunit. The β8-β9 loop (Loop F) showed high KL scores with the β1-β2 and β6-β7 (Pro-loop) loops as well with decreasing distances upon the channel’s opening. Other notable gating mechanisms involved are the pairing of residues from the β1-β2 loop (Loop A) with residues from the strands β1 and β6, as well as the kink of the pore-lining helix. KL divergence proved a valuable tool to filter available data and the novel mechanisms can prove useful both to the academic community that seeks to unravel the complete gating mechanism of GLIC and to the pharmaceutical companies that search for new binding sites within the molecule for new drugs.