Les canaux ioniques sont des complexes macromoléculaires clés exprimés dans tous les types de cellules et sont impliqués dans divers processus physiologiques, y compris la génération et la propagation de potentiels d'action. Des canaux défectueux conduisent à des maladies graves, notamment l'épilepsie, des arythmies et des syndromes douloureux, ce qui en fait une cible potentielle intéressante pour le développement de médicaments. Pour améliorer notre compréhension de ces assemblages biologiques et éventuellement trouver des traitements spécifiques pour les canalopathies, il est crucial d'étudier la structure et la fonction des canaux ioniques. L'objectif principal de cette thèse a été d'étudier ce type de détails structurels et fonctionnels pour trois canaux ioniques associés aux domaines des capteurs de douleur et des canaux potassiques voltage-dépendants en utilisant des techniques de fluorescence et d'électrophysiologie. Dans le premier projet, nous avons étudié la stœchiométrie des canaux hétéromères Kv2.1 / 6.4 (chapitre trois). La technique du décompte de sous-unités isolées (single subunit counting :ssc) permet de compter les sous-unités marquées par fluorescence d’un complexe isolé en déterminant le nombre d'événements de photoblanchiment, qui apparaissent en sauts irréversibles vers le bas sur les traces de fluorescence. Pour désigner la stœchiométrie la plus probable, nous avons utilisé des calculs de probabilités pondérées et avons constaté que les canaux Kv2.1 / 6.4 s'expriment dans un arrangement 2 : 2. Plus précisément, les études fonctionnelles des canaux concatémériques montrent que les sous-unités Kv6.4 et 2.1 doivent être disposées de manière alternée. Le deuxième projet était également basé sur des expériences de SSC et visait à déterminer l'état oligomérique du nouveau canal ionique TACAN (chapitre quatre). Nous avons trouvé une portion significative de canaux intracellulaires, ce qui a provoqué une fluorescence de fond dans les expériences de SSC traditionnelles réalisées avec les cellules mammifères. Pour améliorer le rapport du signal sur bruit de fond, nous avons effectué des expériences de SSC sur des canaux purifiés qui ont été immobilisés sur des lamelles de verre fonctionnalisées Ni-NTA. En utilisant la méthode de calcul décrite dans le premier projet, nous avons trouvé différents états oligomériques et proposons que les canaux TACAN natifs s'assemblent en tétramères qui sont instables lorsqu'ils sont solubilisés dans un détergent. Dans le dernier projet, nous avons étudié la relation structure-fonction de la sous-unité auxiliaire DPP6 pour les canaux Kv4.2 (chapitre cinq). Ici, nous avons progressivement tronqué le grand domaine extracellulaire de 700 acides aminés de DPP6 et étudié son effet sur les courants macroscopiques en utilisant la technique du cut-open voltage clamp. Nous avons constaté que les sous-unités DPP6 avec un domaine extracellulaire court ne parviennent pas à moduler les propriétés du canal aussi efficacement que la DPP6 pleine longueur. Plus précisément, la seconde moitié du domaine extracellulaire b-propeller de DPP6 est responsable d'une inactivation du canal considérablement accélérée. Sur la base de la structure cristalline du domaine extracellulaire, nous avons proposé qu'un domaine b-propeller stable et possiblement la formation de dimères DPP6 sont responsables de la déstabilisation efficace de l'état du canal ouvert. / Ion channels are key macromolecular complexes expressed in all cell types and are involved in various physiological processes including the generation and propagation of action potentials. Defective channels lead to severe diseases including epilepsy, arrhythmias and pain syndromes making them an interesting potential drug target. To improve our understanding of these biological assemblies and eventually find specific treatments for channelopathies, it is crucial to study the structure and function of ion channels. The main purpose of this thesis has been to investigate such structural and functional details of three ion channel complexes from the field of pain sensors and voltage-gated potassium channels using fluorescence and electrophysiological techniques. In the first project, we studied the stoichiometry of heteromeric Kv2.1/6.4 channel complexes (chapter three). Single subunit counting (SSC) allows to directly count the number of fluorescently labeled subunits by determining the number of irreversible, step-wise photobleaching events. To determine the most probable stoichiometry, we used weighted likelihood calculations and found that Kv2.1/6.4 channels express in a 2:2 arrangement. More precisely, functional studies of concatemeric channels (performed by our collaborators) illustrate that Kv6.4 and 2.1 subunits need to be arranged in an alternating fashion. The second project was also based on SSC experiments and aimed at determining the oligomeric state of the novel ion channel TACAN (chapter four). We found a significant amount of channels in the intracellular which caused background fluorescence in traditional SSC experiments performed in cells. To improve the signal to background ratio, we performed SSC experiments on purified channels that were immobilized on Ni-NTA functionalized glass coverslips. Using the model selection method described in the first project, we found different oligomeric states and propose that native TACAN channels assemble as tetramers which are unstable when solubilized in detergent. In the last project, we investigated the structure-function relation of the auxiliary DPP6 subunit in Kv4.2 channel complexes (chapter five). Here, we progressively truncated DPP6’s 700 amino acids long extracellular domain and studied its effect on macroscopic currents using the cut-open voltage clamp technique. We found that DPP6 subunits with a short extracellular domain fail to modulate the channel properties as efficiently as the full length DPP6. More precisely, the second half of the extracellular b-propeller domain of DPP6 is responsible for drastically accelerated channel inactivation. Based on the crystal structure of the extracellular domain, we proposed that a stable b-propeller domain and possibly DPP6 dimer formation is responsible for destabilizing the open channel state efficiently.

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.

Voltage gated potassium channels (Kv) play a key role in the nervous system- not only due to their involvement in the action potential. Vertebrates express four subtypes, which are termed Kv1, Kv2, Kv3 and Kv4, respectively. Tsha3 is a Kv1 channel which was originally isolated from brain tissue of rainbow trout (Oncorhynchus mykiss). This channel possesses an unique amino terminus and a characteristic amino acid sequence in the T1 domain, which is engaged in the oligomerization of Kv α-subunits and is thus involved into the segregation of subfamilies. The two major goals of this thesis were the structural and functional characterization of the N-terminal cytosolic domain of Tsha3 as well as the invention of a system to gain data about the functional dynamics of full length Kv channels. Molecular biological techniques were used to isolate mRNA from trout brains, to transcribe it into cDNA and clone it into vectors. DNA from such plasmids was ligated into expression vectors for heterologous expression in E. coli, P. pastoris and Sf21 cells, with concomitant fusion of marker proteins (GFP or DsRed) or tags (6 x HisTag or StrepTagII) due to the individual experiment. Protein was overexpressed in E. coli and affinity purified to analyze separated domains with biochemical (SDS-PAGE and Western Blot, Pull-Down-Assay or Dot-Blot-Assay) or biophysical (CD-spectroscopy, EPR spectroscopy) efforts. The P. pastoris system to express Tsha1 was established, to generate a system for future EPR-measurements of whole Kv channels. Heterologous expression of Kv1α (Tsha3 and Tsha1) and the core domain of Kvβ in Sf21 cells was performed to analyze the subcellular distribution of the respective subunits via fluorescence microscope and via subcellular fractionation of cell lysates with downstream biochemical analyses (SDS-PAGE and Western Blot). Furthermore the gating of diverse fusion constructs of Tsha3 in co-expressions and the gating of diverse cystein substitution mutants of Tsha1 were measured via path-clamp recordings in whole cell modus. The structural analyses of the N-terminal cytosolic domain (NCD) of Tsha3 revealed that the 128 amino acid containing part before the T1-domain (Tsha3-NT) can be structurally divided into three parts of different structure and mobility. The most outward part possesses a very high mobility and is putatively unfolded as random coil. This section is expected to express no tertiary contacts. The middle part of Tsha3-NT is structured in α-helices and β-sheets and thus slightly immobile. This folded part is also assumed to build no tertiary structure and to be exposed into the cytosol. The third, which is directly neighboring the T1 domain, has the most restricted mobility of Tsha3-NT. It consists predominantly of α-helices and exhibits a tertiary structure, putatively with the T1 domain. Tsha3-NCD self-tetramerizes and oligomerizes with Tsha1, although mutations exist in Tsha3 in conserved amino acids, which were reported to function in subfamily specific hetero-tetramerization. Thus it is proven, that Tsha3 takes part in the segregation into the Kv1 subfamily. Furthermore, Tsha3 interacts with the core domain of Kvβ2 although there are also mutations in the reported consensus sequence for interaction. Association of Kvβ2 in co-expression studies directs Tsha3-DsRed fusion constructs from internal vesicular structures into the cell membrane. But the fusion with DsRed is leading to a loss of function of Tsha3 which cannot be rescued by co-expression of the chaperone Kvβ2. But- without fusion of marker proteins- Tsha3 was identified as an outward rectifier in a cooperative Bachelor Thesis. These structural data lead to the assumption, that Tsha3-NT exhibits lateral interactions and especially the helical but mobile middle part of the N-terminus can play such a role. Due to the localization next to the membrane, interactions with membrane proteins- putatively with protein cascades are possible. Although Tsha3-NT contains no reported interaction domains for protein-protein interactions, follow-up experiments should be performed to shed light on this interesting question. Tsha1 C30S C31S C180S C224A C239S C389S C424S C476S is a complete cysteine free mutant, which was identified as a functional voltage-gated potassium channel. It was expressed in and purified from eukaryotic cells (P. pastoris) and therefore it can be assumed to be properly folded and modified. After a slight optimization of the features of expression, this system can be used to reconstitute Tsha1 channels into liposomes and use them for Freeze Quench EPR to gain structural information about a Kv1 channel in the open as well as in the closed state. This is the first report of the establishment of a full length Kv for studies of structure and functional dynamics experiments.

Les canaux ioniques sont des protéines membranaires permettant le transport ionique au travers des membranes biologiques. Du fait de leur omniprésence dans l'organisme, ils représentent une classe de cibles thérapeutiques encore actuellement peu exploitée du fait de limitations expérimentales dans leur étude. La mesure électrique de l'activité des canaux ioniques au sein de bicouches biomimétiques reconstituées in vitro permettrait de répondre à ces limitations. Cependant, il n'existe actuellement pas de système satisfaisant au cahier des charges complet pour de telles analyses : stabilité et pureté de la bicouche, faible niveau de bruit, insertion rapide des canaux ioniques, intégration dans un dispositif fluidique, possibilité de mener une caractérisation optique simultanée. L'objectif de ces travaux de thèse était d'évaluer dans quelle mesure l'utilisation d'un substrat SOI (Silicon On Insulator) comprenant des nanopores pourrait permettre de répondre à tous ces critères. Des nanopores de diamètre compris entre 10 nm et 160 nm ont été réalisés à partir d'un substrat SOI. Une cellule fluidique transparente est utilisée pour l'adressage fluidique. Cette cellule permet d'autre part la double caractérisation électrique et optique. Les propriétés électriques en milieu liquide du dispositif ont été étudiées et permettent de dégager des perspectives d'amélioration. La double caractérisation électrique et optique est démontrée au moyen d'expériences de capture de nanoparticules fluorescentes sur les nanopores. Enfin, des premiers résultats prometteurs d'obtention d'une bicouche lipidique suspendue sont présentés. / Ion channels are membrane proteins responsible for ion transport across biological membranes. Due to their ubiquity, they are promising drug targets but are not yet fully exploited as such due to experimental restrictions in their study. Electrical measurement of ion channels activity within in vitro artificial lipid bilayers would enable to overcome these restrictions. However, there is not yet a system satisfying all the requirements for ion channels studies: stability and purity of the lipid bilayer, low noise level, fast insertion of ion channels, fluidic integration, ability to perform simultaneous optical characterization. The aim of this phD was to assess in which extent the use of an SOI (Silicon On Insulator) substrate bearing nanopores could satisfy all these requirements. 10 nm to 160 nm diameter nanopores were fabricated in an SOI substrate and characterized. A transparent fluidic cell was used for fluidic addressing. This transparent cell allows combined electrical and optical characterization. Electrical properties of the device in aqueous environment were studied, allowing to bring out improvement prospects. The combined electrical and optical characterization was demonstrated with fluorescent nanoparticle trapping experiments on the nanopores. Finally, promising results about the formation of a free-standing lipid bilayer are presented.

Indiana University-Purdue University Indianapolis (IUPUI) / Regulation of coronary blood flow is a highly dynamic process that maintains the delicate balance between oxygen delivery and metabolism in order to preserve cardiac function. Evidence to date support the finding that Kv and Cav1.2 channels are critical end-effectors in modulating vasomotor tone and blood flow. Yet the role for these channels in the coronary circulation in addition to their interdependent relationship remains largely unknown. Importantly, there is a growing body of evidence that suggests obesity and its pathologic components, i.e. metabolic syndrome (MetS), may alter coronary ion channel function. Accordingly, the overall goal of this investigation was to examine the contribution coronary Kv and Cav1.2 channels to the control of coronary blood flow in response to various physiologic conditions. Findings from this study also evaluated the potential for interaction between these channels, i.e. electromechanical coupling, and the impact obesity/MetS has on this mechanism. Using a highly integrative experimental approach, results from this investigation indicate Kv and Cav1.2 channels significantly contribute to the control of coronary blood flow in response to alterations in coronary perfusion pressure, cardiac ischemia, and during increases in myocardial metabolism. In addition, we have identified that impaired functional expression and electromechanical coupling of Kv and Cav1.2 channels represents a critical mechanism underlying coronary dysfunction in the metabolic syndrome. Thus, findings from this investigation provide novel mechanistic insight into the patho-physiologic regulation of Kv and Cav1.2 channels and significantly improve our understanding of obesity-related cardiovascular disease.

<p> We developed some universal platforms to overexpress the minimal functional entities of ion channels. The modular property of ion channels have been demonstrated from many aspects, such as crystal structures, chimeric channel experiments and discovery of similar modules in distantly related protein families. Thus it should be feasible to express each module independent of other channel modules. The pore-forming module of ion channels has multiple important properties as selectivity, conductivity and drug-binding. If it can be overexpressed, it will provide valuable information about channel selectivity to different ions and structural bases for drug binding as well as important application in drug screening and rational drug design. </p> <p> To test this, we first used the model channel KcsA to identify the minimal requirements for a pore-forming domain to functionally exist independently. Chapter 2 of this thesis explains in detail how the wild type C-terminal cytoplasmic domain of KcsA functions. We found that this domain has dual function as pH-sensor and tetramerization domain, and it is essential for the expression of the pore-forming domain of KcsA. Once we knew the physiological role of the cytoplasmic domain, the scenario was set to answer the question of how to make it better for the application of structural and functional studies. </p> <p> In chapter 3 and chapter 4, we replaced the wild type C-terminal domain with non-native tetramerization domains. We identified the direct correlation between protein expression level and overall thermostability of pore-forming domains. The C-terminal tetramerization domains stabilize channels in a cooperative way and play a critical way in in vivo channel assembly. The selection of the linker between pore-forming domain and tetramerization domain, the splicing motif, and the handedness of C-terminal tetrameric coiled coils all affect channel expression level and stability. </p> <p> We applied our finding in KcsA to a wide range of ion channels in chapter 5, including voltage-gated potassium channels, Ca2+-gated potassium channels, inwardrectifying potassium channels, cyclic nucleotide-gated potassium channels and voltagegated sodium channels. We managed to express similar minimal structural modules from these more structurally complicated channels with the assistance of different cytoplasmic tetramerization domains. Several minimal channels expressed well and showed similar biophysical and functional property as the wild type channels. </p> <p> These studies demonstrate that the pore-forming modules of ion channels can be expressed independently while retaining the proper structure and drug-binding properties as their wild type predecessors when using our universal expression platform. The potential application in structural studies and drug-screening is promising. </p> / Thesis / Doctor of Philosophy (PhD)

No description available.

Ionenkanäle spielen eine wichtige Rolle in vielen physiologischen Prozessen. Während manche Kanäle hochspezifisch für eine Ionensorte sind, sind andere Kanäle weniger selektiv. Tetramere Ionenkanäle weisen eine gemeinsame Grundarchitektur auf, von der nur ionotrope Glutamatrezeptoren (iGluRs) abweichen, deren Struktur im Vergleich zu den anderen invertiert ist. Eine Untergruppe der iGluRs stellen α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid Rezeptoren (AMPARs) dar, welche im Zentralnervensystem von Wirbeltieren die Mehrheit der exzitatorischen Neurotransmission übernehmen. Eine einzelne posttranskriptionelle Veränderung (Glutamin zu Arginin) an der Spitze des Selektivitätsfilters (SF, Q/R Stelle) von AMPAR-Typ 2 (GluA2) macht den Kanal quasi undurchlässig für Ca2+. Das Ziel dieser Arbeit war das Erstellen einer GluA2-Kanalchimäre mit Hilfe des bakteriellen Kanals NaK. Mutation rund um die Q/R Stelle wurden nicht toleriert von der Chimäre, während C-terminale Mutationen des SF stabil waren und für strukturelle Studien verwendet wurden. Die Entfernung einer Aminosäure im Vergleich zum NaK Wildtyp erzeugte eine Begradigung des SF und eine Erweiterung der wassergefüllten Ausbuchtung. Überraschenderweise kristallisierten die NaK Chimären überwiegend in zweifach symmetrischer Anordnung. Vor allem im SF kam es zu ausgeprägter lokaler Asymmetrie, unabhängig von der Ionenzusammensetzung. Um die Flexibilität des SF näher zu untersuchen, wurden Aminosäuren von NaK in GluA2 integriert und mithilfe von Patch-clamp Elektrophysiologie untersucht. Gleichsam wie in NaK, wurden Mutationen an der Filterspitze nicht toleriert, wohingegen die C-terminale Hälfte problemlos ausgetauscht werden konnte. Die funktionelle Integrität der NaK Chimäre wurde mithilfe von Einzelkanalmessungen in Bilayern überprüft. Zusätzlich wurden, auf Basis der gelösten Strukturen, Molekulardynamiksimulationen durchgeführt, welche einen dynamischen Einblick in den Permeationsmechanismus erlauben. / Ion channels play an important role in many physiological processes, for example the generation of action potentials. While some channels display high selectivity for one ion species, others are more promiscuous. All tetrameric cation channels share the same principal architecture, but the transmembrane domain of ionotropic glutamate receptors (iGluRs) is inverted relative to the other members. A subfamily of iGluRs, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), mediates the vast majority of excitatory neurotransmission in the vertebrate central nervous system. In AMPA-type 2 iGluRs (GluA2), a single post-transcriptional modification (glutamine to arginine) at the tip of the selectivity filter (SF, Q/R site) renders the channel Ca2+ impermeable. The aim of this thesis was therefore to create an AMPAR pore mimic by using the bacterial channel NaK. Unfortunately, mutations around the Q/R site abolished expression of the NaK-GluA2 chimera, while C-terminal mutations of the SF were stable and could be used for structural studies. The shortening of the SF by one amino acid caused a straightening and opened an extended water-filled vestibule. Strikingly, most of the tested chimeras exhibited twofold symmetry with strong local asymmetry in the mutated part of the SF independent of the ion type present. For functional tests, residues from NaK wt were also swapped into GluA2. N-terminal mutations abolished the current response, whereas C-terminal mutations behaved wt-like. Single-channel bilayer experiments confirmed the functional integrity of the NaK-GluA2 chimera. Additionally, extensive molecular dynamics simulations, based on the solved structures, were carried out alongside. In summary, it could be demonstrated that the SF of the non-selective NaK-GluA2 chimera is highly flexible and accommodated all tested ions. Ion binding is accompanied by local asymmetric rearrangements, possibly creating an energetically simple way to allow permeation.

Ionenkanäle sind für die zelluläre Homöostase und die elektrische Aktivität in höheren Eukaryoten essentiell. Die vorliegende Arbeit widmet sich dem nichtselektiven Kanal NaK und seinen kaliumselektiven Mutanten. Die Bedeutung von Ionenkanälen wird in Kapitel 1 speziell für die kationenselektive Ionenkanal-Superfamilie diskutiert. Darin werden verschiedene Vertreter dieser Superfamilie untersucht und ihre Strukturen und Ionenselektivität analysiert. In Kapitel 2 wird gezeigt, dass NaK zwei unterschiedliche Selektivitätsfilterkonformationen aufweist, die entweder durch Na+- oder K+-Ionen stabilisiert sind. Unter Verwendung von Festkörper-NMR Spektroskopie und molekulardynamischen Simulationen wurden zwei Ionenleitungswege entdeckt. In Kapitel 3 wurde eine Kristallstruktur von NaK ermittelt, welche die vorhergesagte und für den Seiteneintrittsmechanismus essentielle seitliche Ionenbindungsstelle bestätigt. Die zwei Untereinheiten in der asymmetrischen Einheit zeigen die dynamische Natur der unteren Teile der Transmembranhelices sowie duale Konformationen für die Reste im Selektivitätsfilter. Im Gegensatz zu NaK sind die kaliumselektiven Mutanten ionensensitiver, wie in Kapitel 4 gezeigt: Unter Na+-Bedingungen verliert der gesamte Selektivitätsfilter in den kaliumselektiven Mutanten seine Stabilität. Die stärkere Verbindung zwischen Selektivitätsfilter und der Porenhelix in den kaliumselektiven Mutanten ermöglicht keine nichtselektive Ionenleitung. Unter Verwendung von protonendetektierter Festkörper-NMR wurde die Wechselwirkung zwischen Wassermolekülen und der kaliumselektiven Mutante NaK2K charakterisiert und präsentiert in Kapitel 5. Es wurde gezeigt, dass der Selektivitätsfilter von NaK2K unter physiologischen Bedingungen wasserfrei ist. Diese Ergebnisse werden in Kapitel 6 im Ganzen betrachtet und die verbleibenden Fragen werden erörtert, außerdem wird ein kurzer Ausblick auf die zukünftige Forschung zum Thema Ionenselektivität im NaK-Kanal gegeben. / Ion channels are essential to cellular homeostasis and electrical activity in higher eukaryotes. This thesis discusses the non-selective channel NaK and its potassium-selective mutants. The importance of ion channels is discussed in chapter 1 with a special focus on the tetrameric cation-selective ion channel superfamily. Various members of this superfamily are explored and their structures and ion selectivity are analysed. NaK is shown to have two distinct selectivity filter conformations that are stabilized by either Na+ or K+ ions in chapter 2. Using solid-state NMR spectroscopy and molecular dynamics simulations, two ion conduction pathways were discovered. In chapter 3 a crystal structure of NaK was determined that confirms the previously predicted side-entry ion binding site, essential to the side-entry pathway. The two subunits in the asymmetric unit display the dynamical nature of the lower parts of the transmembrane helices as well as dual conformations for residues in the selectivity filter. In contrast to NaK the potassium-selective mutants are more ion sensitive as shown in chapter 4. The entire selectivity filter loses its stability under Na+ conditions for the potassium-selective mutants. The stronger connection of the selectivity filter and the pore helix in the potassium-selective mutants does not allow for non-selective ion conduction. Using proton-detected ssNMR, the interaction between water molecules and the potassium-selective mutant NaK2K was characterized and this is presented in chapter 5. The selectivity filter of NaK2K was shown to be free of water under physiological conditions. These results get put in perspective and the questions which remain are discussed in chapter 6. A short outlook on future research for the topic of ion selectivity in the NaK channel is given.

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>