Pentameriska ligandstyrda jonkanaler (pLGICs) är avgörande för omvandlingen av kemisk till elektrisk signalöverföring i djurs nervsystem. Dysfunktion i dessa kanaler har visat sig vara kopplad till flera sjukdomar inklusive epilepsi, schizofreni, Alzheimers och autism, vilket gör dem till en måltavla för en mängd olika läkemedel. Att studera eukaryota kanaler är dock mycket utmanande, så upptäckten av prokaryota homologer, som är mycket lättare att studera, har därmed bidragit mycket till förståelsen för struktur och funktion hos proteiner i denna familj. I detta projekt producerades och renades en prokaryotisk pLGIC kallad DeCLIC från Escherichia coli. Strukturell bestämning av kanalen genomfördes med användning av kryo-elektronmikroskopi vid lågt pH och i närvaro av kalcium. En elektrontäthet med 3.4 Å upplösning uppnåddes och jämfördes med tidigare bestämda strukturer vid olika förhållanden i ett försök att bestämma hur proteinets struktur moduleras av kalcium och pH. Resultaten visar flera skillnader i kanalens konformation i närvaro och frånvaro av kalcium såväl som vid olika pH-värden. Dessutom antyder analys av den bestämda elektrontätheten ett möjligt intermediärt tillstånd vid lågt pH i närvaro av kalcium. / Pentameric ligand-gated ion channels (pLGICs) are crucial for the conversion of chemical to electrical signaling in the nervous system of mammals. Dysfunction in these channels has been found to be connected to several diseases including epilepsy, schizophrenia, Alzheimer’s, and autism, making them the target of a wide variety of therapeutic agents. However, studying eukaryotic channels is challenging so the discovery of prokaryotic homologs that are much easier to study has thus greatly helped in the understanding of the structure and function in this family of proteins. In this project, a prokaryotic pLGIC called DeCLIC was produced and purified from Escherichia coli. Structural determination of the channel was pursued using cryo-electron microscopy at a low pH and in the presence of calcium. An electron density at 3.4 Å resolution was achieved and compared to previously determined structures at different conditions in an attempt to determine the structural modulation of calcium and pH. Results show multiple differences in channel conformation in the presence and absence of calcium as well as in different pH conditions. Furthermore, analysis of the determined electron density suggests a possible intermediate state at low pH in the presence of calcium.

Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane. / <p>QC 20151217</p>

Ligand-gated ion channels play an important role in electrochemical signal transduction across diverse organisms, yet their structural and functional intricacies are not fully understood. Particularly lacking is the knowledge of their response to variations in pH, an aspect necessary for understanding their physiological relevance and potential therapeutic targeting in neurological diseases. In this thesis project, I have investigated the mechanistic response of sTeLIC, a recently reported prokaryotic member of the pentameric ligand-gated ion channel family, to different environmental conditions. Using molecular dynamics simulations, a total of 16 different environmental conditions have been explored including variations in pH (neutral and alkaline), the presence and absence of calcium, and the inclusion of an electric field acting as an external driving force on charged atoms. The results reveal a comprehensive pH-sensing and gating mechanism involving key residues, notably E106 (on the β6 strand) and E160 (on loop F), and their local microenvironments. Additionally, an inhibitory mechanism for calcium is proposed, with E160 playing an important role. The simulations including an electric field has provided support for a non-conventional ion pathway through the pore. Collectively, these results offer insights into a mechanistic framework that may extend to other physiologically relevant systems, providing a foundation for further investigations and potential future therapeutic intervention. / pLGICs har en viktig roll i det elektrokemiska signalsystemet i många organismer, men detaljerna i deras struktur och framför allt funktion är fortfarande inte helt klargjorda. Särskilt är detaljerna kring deras reaktion på ändringar i pH-värde relativt okända, vilket är en viktig del i att förstå kanalernas fysiologiska roll och för att potentiellt hitta läkemedel mot neurologiska sjukdomar där dessa är inblandade. I det här arbetet har jag undersökt hur sTeLIC, som är ett nyligen publicerat bakteriellt protein i familjen pLGICs, reagerar på olika ändringar i miljön. Jag har använt molekyldynamiksimuleringar för att unders öka 16 olika miljöer med två olika pH-värden (neutralt och alkaliskt), med eller utan kalcium samt med eller utan en extern drivkraft över membranet i form av ett elektrisk fält. Arbetet har resulterat i en föreslagen mekanism förhur sTeLIC känner av pH och hur öppningen av kanalen går till. Denna mekanism involverar aminosyrorna E106, som finns på β6-strängen, och E160, som finns på F-loopen, samt deras omgivning. Dessutom har en modulatorisk mekanism föreslagits för en kalciuminhiberande effekt på sTeLIC som också involverar E160. Simuleringarna med en drivkraft över membranet har gett stöd för en ny väg för joner genom kanalen. Tillsammans ger dessa resultat insikt i en mekanism som eventuellt kan appliceras p ̊a andra system. Detta har lagt grunden för fortsatt undersökning som potentiellt kan leda till framtida läkemedelsutveckling inom området.