Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "December 1998" Typescript. Vita. Includes bibliographical references (l. 140-148). Also available on the Internet.

Thesis (Ph.D.)--Indiana University, 2010. / Title from screen (viewed on April 1, 2010). Department of Pharmacology and Toxicology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Theodore R. Cummins, Grant D. Nicol, Gerry S. Oxford, Andy Hudmon, John H. Schild. Includes vitae. Includes bibliographical references (leaves 232-266).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 68-82).

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 134-139.

Excitation-contraction (EC) coupling is the process by which Ca²⁺ is released from the sarcoplasmic reticulum (SR) and is fundamental to cardiac and skeletal muscle function. The SR contains many uncharacterised ion channels and proteins which may influence EC coupling and in this thesis I have investigated the biophysical properties of some of these channels. I have demonstrated that the single-channel gating and conducting properties of SR K⁺ channels from various mammalian species (rabbit, sheep and mouse) are very similar. I investigated the actions of possible physiological regulators of these channels and demonstrated that luminal Ca²⁺ and Mg²⁺ can block K⁺ flux in a voltage-dependent manner, while luminal Ca²⁺, Ni²⁺, and alkaline pH can reduce Po by additional mechanisms. I also characterised the single-channel properties of the various SR anion channels that are observed after incorporating mammalian SR vesicles into artificial membranes. The trimeric intracellular cation channels (TRIC-A and TRIC-B) and Mitsugumin 23 (MG23) are suggested to be SR cation channels. I have therefore utilised Tric-a KO and Mg23 KO mice to study SR membranes devoid of TRIC-A and MG23. Additionally, I have begun to investigate the single-channel properties of purified c. elegans TRIC-B1 and human TRIC-A. I found that SR K⁺ channel function was altered in SR from Tric-a KO or Mg23 KO tissue, however the underlying mechanisms for the observed changes appear to be complex. My initial studies of the purified TRIC-A and TRIC-B proteins show that they are permeable to K⁺, Ca²⁺, choline, and Cl^-, properties which deviate from those of SR K⁺ channels from rabbit, mouse and sheep. This may reflect species differences or alterations to protein function caused during the purification process or that SR K⁺ channels remain an unidentified class of ion channel.

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.

Sodium ion (Na+) channels play an important role in excitable cells, as they are responsible for the initiation of action potentials. Understanding the electrical characteristics of sodium channels is essential in predicting their behavior under different physiological conditions. We investigated several Markov models for the human cardiac sodium channel (NaV1.5) to derive a minimal mathematical model that can describe the reported experimental data obtained using major voltage-clamp protocols. We obtained simulation results for current-voltage relationships, steady-state inactivation, the voltage dependence of normalized ion channel conductance; activation and deactivation, fast and slow inactivation and recovery from inactivation kinetics. Good agreement with the experimental data provides us with the mechanisms of the fast and slow inactivation of the human sodium channel and the coupling of its inactivation states to the closed and open states in the activation pathway.

Dissociated neuronal cell cultures grown on substrate integrated microelectrode arrays (MEAs) generate spontaneous activity that can be recorded for up to several weeks. The signature wave shapes from extracellular recording of neuronal activity display a great variety of shapes with triphasic signals predominating. I characterized extracellular recordings from over 600 neuronal signals. I have preformed a categorical study by dividing wave shapes into two major classes: (type 1) signals in which the large positive peak follows the negative spike, and (type 2) signals in which the large positive peak precedes the negative spike. The former are hypothesized to be active signal propagation that can occur in the axon and possibly in soma or dendrites. The latter are hypothesized to be passive which is generally secluded to soma or dendrites. In order to verify these hypotheses, I pharmacologically targeted ion channels with tetrodotoxin (TTX), tetraethylammonium (TEA), 4-aminopyridine (4-AP), and monensin.

The prokaryotic Erwinia chrysanthemi ligand-gated ion channel (ELIC) is competitively inhibited by acetylcholine (Pan et al., 2012). Acetylcholine is the native agonist of the structurally related family of eukaryotic acetylcholine receptors, which like ELIC are pentameric ligand-gated ion channels. To understand the opposite effect upon acetylcholine binding between ELIC and acetylcholine receptors, we used statistical coupling analysis to predict mutations necessary for installing acetylcholine agonism into ELIC. Statistical coupling analysis was performed on the acetylcholine binding protein from Lymnaea stagnalis. This protein is a structural surrogate for the agonist binding domain of acetylcholine receptors, for which a high-resolution structure in complex with acetylcholine is available. Our analysis identified a group of statistically coupled residues that comprises several amino acids previously implicated in acetylcholine agonism of acetylcholine receptors. Mapping these residues onto ELIC revealed 15 residue discrepancies, 4 of which were chosen for initial mutagenesis based upon their proximity to the known agonist binding site. Electrophysiological characterization of ELIC mutants indicates that the potency of the native agonist, cysteamine, is decreased, highlighting the optimized role wild-type residues serve in native agonism. None of the mutants were activated by acetylcholine, however the double mutant A75D/F133W abolished competitive antagonism by acetylcholine, and instead led to acetylcholine dependent potentiation of cysteamine-induced currents. This work demonstrates the ability of statistical coupling analysis to identify functionally important residues in pentameric ligand-gated ion channels and reveals that acetylcholine can be converted from a competitive antagonist into a potentiator, by installing two residues present in acetylcholine receptors.

MicroRNA-9 (miR-9) modulates gene expression and demonstrates high structural conservation and wide expression in the central nervous system. Bioinformatics analysis predicts almost 100 ion channels, membrane transporters and receptors, including genes linked to primary familial brain calcification (PFBC), as possible miR-9-5p targets. PFBC is a neurodegenerative disorder, characterized by bilateral and symmetrical calcifications in the brain, associated with motor and behavioral disturbances. In this work, we seek to study the influence of miR-9-5p in regulating genes involved in PFBC, in an osteogenic differentiation model with SaOs-2 cells. During the induced calcification process, solute carrier family 20 member 2 (SLC20A2) and platelet-derived growth factor receptor beta (PDGFRB) were downregulated, while platelet-derived growth factor beta (PDGFB) showed no significant changes. Significantly decreased levels of SLC20A2 and PDGFRB were caused by the presence of miR-9-5p, while PDGFB showed no regulation. We confirmed the findings using an miR-9-5p inhibitor and also probed the cells in electrophysiological analysis to assess whether such microRNA might affect a broader range of ion channels, membrane transporters and receptors. Our electrophysiological data show that an increase of the miR-9-5p in SaOs-2 cells decreased the density and amplitude of the output ionic currents, indicating that it may influence the activity, and perhaps the expression, of some ionic channels. Additional investigations should determine whether such an effect is specific to miR-9-5p, and whether it could be used, together with the miR-9-5p inhibitor, as a therapeutic or diagnostic tool.