<p> Potassium channels allow selective flow of K+ ions across impermeable membranes. They switch between closed and open states in response to stimulus. The closed state of KcsA, a potassium channel from the bacteria streptomyces lividans, has been crystallized and studied. However, attempts to obtain a structural description of gating transition of the channel have been hampered because the open state is transiently occupied. In this study, we investigated changes in gating functions of KcsA caused by variations in environmental parameters such as pH, voltage and also mutation in KcsA using planar bilayer and patch clamp channel recording techniques to improve our understanding on the functional aspect of gate activation of KcsA.</p> / Thesis / Master of Science (MSc)

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

No description available.

Understanding protein-ligand interactions is highly important in drug development. In the present work the objective is to comprehend the link between structure and function using molecular modelling. Specifically, this thesis has been focused on implementation of receptor flexibility in molecular docking and studying structure-activity relationships of potassium ion channels and their blockers. In ligand docking simulations protein motion and heterogeneity of structural waters are approximated using an ensemble of protein structures. Four methods of combining multiple target structures within a single grid-based lookup table of interaction energies are tested. Two weighted average methods permit consistent and accurate ligand docking using a single grid representation of the target protein structures. Quaternary ammonium ions (QAIs) are well known K+ channel blockers. Conformations around C–N bonds at the quaternary centre in tetraalkylammonium ions in water solution are investigated using quantum mechanical methods. Relative solvation free energies of QAIs are further estimated from molecular dynamics simulations. The torsion barrier for a two-step interconversion between the conformations D2d and S4 is calculated to be 9.5 kcal mol–1. Furthermore D2d is found to be more stable than the S4 conformation which is in agreement with experimental studies. External QAI binding to the K+ channel KcsA is also studied. Computer simulations and relative binding free energies of the KcsA complexes with QAIs are calculated. This is done with the molecular dynamics free energy perturbation approach together with automated ligand docking. In agreement with experiment, the Et4N+ blocker in D2d symmetry has better binding than the other QAIs. Binding of blockers to the human cardiac hERG potassium channel is studied using a combination of homology modelling, automated docking and molecular dynamics simulations. The calculations reproduce the relative binding affinities of a set of drug derivatives very well and indicate that both polar interactions near the intracellular opening of the selectivity filter as well as hydrophobic complementarity in the region around F656 are important for blocker binding. Hence, the derived model of hERG should be useful for further interpretations of structure-activity relationships.

Die vorliegende Arbeit untersucht die Struktur der Untereinheit B des natriumabhängigen Kplus-Aufnahmesystems KtrAB aus Vibrio alginolyticus. Sie gehört zu einer Superfamilie von Kaliumtransportproteinen, die auf der im KcsA-Kanal gefundenen MPM-Topologie beruht. Eine MPM-Domäne besteht aus zwei Transmenbranhelices, die durch einen in die Membran zurückgefalteten P-Loop verbunden sind. Im KcsA-Kanal arrangieren sich vier dieser Untereinheiten um eine zentrale Pore. Im Unterschied zum KcsA-Kanal aus S. lividans sind in dieser Superfamilie die MPM-Domänen durch cytoplasmatische Loops miteinander verbunden. Die KtrB-Topologie beruht zur Zeit nur auf Modellen und wird in dieser Arbeit genauer untersucht. Dabei konnte die MPM-Faltung mittels PhoA-Fusionen für die Bereiche B-D von VaKtrB bestätigt, und die sogenannte Turret -Struktur, wie sie im KcsA-Kanal gefunden wurde, nachgewiesen werden. Bei Modellierungstudien der KtrB-Topologie fiel im Besonderen die M2C-Helix auf, woraufhin Durell und Guy drei Unterteilungen für diesen Bereich vorschlugen: M2C1: alpha-Helix; M2C2: flexibler Bereich; M2C3: teilweise amphiphile Helix. Es herrschen zwei Modelle dieser Helix vor, wobei sie sich vor allem in der Lokalisation von M2C2 und M2C3 unterscheiden. Im ersten bildet M2C2 einen gestreckten Übergang von M2C1 zu M2C3, die waagerecht in die Membranoberfläche eingelagert ist. Im 2. Modell liegt M2C2 innerhalb der Kavität als Schleife vor und M2C3 steckt senkrecht in der Membranoberfläche. Die in dieser Arbeit gewonnenen Daten (Cysteinzugänglichkeit für Maleimide, PhoA-Fusionen, ESR-Spektroskopie) erhärten das 1. Modell und unterstützen die These, dass M2C2 einen flexiblen Bereich innerhalb von M2C bildet, der wichtig für den Transport bzw. dessen Regulation ist. Die Faltung der M2C-Helix konnte allerdings nicht abschließend geklärt werden. Desweiteren deuten die Daten dieser Arbeit auf eine wässrige Verbindung bis tief in KtrB vom Periplasma her hin.

No description available.

<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)