Wong, Tak Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 96-104). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Chinese Abstract --- p.v / Acknowledgements --- p.vii / Abbreviation --- p.ix / Table of Content --- p.xi / List of figures --- p.xiv / List of tables --- p.xv / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Problem of soil salinization and sodification: reducing crop productivity --- p.1 / Chapter 1.2 --- Effects of high salinity on plant growth --- p.2 / Chapter 1.2.1 --- Ion toxicity --- p.2 / Chapter 1.2.2 --- Osmotic stress --- p.3 / Chapter 1.2.3 --- Oxidative stress --- p.3 / Chapter 1.3 --- Overview of salt tolerance mechanisms in plant --- p.4 / Chapter 1.3.1 --- Maintenance of ion homeostasis --- p.4 / Chapter 1.3.2 --- Maintaining osmotic homeostasis --- p.5 / Chapter 1.3.3 --- Detoxification of Reactive oxygen species --- p.5 / Chapter 1.4 --- The important role of CI- in plant salt stress tolerance research --- p.6 / Chapter 1.5 --- Introduction to chloride channel (CLC) family --- p.7 / Chapter 1.6 --- E. coli CLC-ecl: The first CLC member found to function as antiporter --- p.8 / Chapter 1.7 --- Yeast GEF1: eukaryotic model for early plant CLC complementation studies --- p.9 / Chapter 1.8 --- Mammalian CLC family: 4 channels and 5 antiporters --- p.10 / Chapter 1.8.1 --- CLC-4 and -5: First eukaryotic CLC member found to be function as antiporter --- p.13 / Chapter 1.8.2 --- CLC-7 function as antiporter and regulate lysosomal acidification --- p.13 / Chapter 1.8.3 --- "CLC-6 select nitrate over chloride, unlike other mammalian CLC members" --- p.14 / Chapter 1.9 --- Introduction to Plant CLC members --- p.14 / Chapter 1.10 --- Tobacco CLC-Ntl co-localized with mitochondrial markers in plant and may cause current on Xenopus oocytes membrane --- p.15 / Chapter 1.11 --- Rice CLCs may involved in salt tolerenace and growth regulation --- p.16 / Chapter 1.12 --- Arabidopsis CLC members are extensively studied --- p.18 / Chapter 1.12.1 --- AtCLCa regulates nitrate accumulation --- p.20 / Chapter 1.12.2 --- "AtCLCb, a nitrate/proton antiporter with unclear physiological role" --- p.22 / Chapter 1.12.3 --- "AtCLCc selective chloride over nitrate, involved in salt tolerance" --- p.23 / Chapter 1.12.4 --- AtCLCd and AtCLCf both localized on Golgi network --- p.25 / Chapter 1.12.5 --- AtCLCe may regulate ionic strength of chloroplast thylakoid membrane --- p.26 / Chapter 1.13 --- Previous work in Prof. Lam's laboratory --- p.26 / Chapter 1.14 --- "Reason, Hypothesis, Objective and long term significance" --- p.28 / Chapter 2. --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- "Bacterial strains, animals, plants and plasmid vectors" --- p.30 / Chapter 2.1.2 --- Chemicals and Enzymes --- p.33 / Chapter 2.1.3 --- Commercial kits --- p.33 / Chapter 2.1.4 --- Primers --- p.35 / Chapter 2.1.5 --- Equipments and facilities used --- p.36 / Chapter 2.1.6 --- "Buffer, solution, gel and medium" --- p.36 / Chapter 2.1.7 --- Software --- p.36 / Chapter 2.2 --- Methods --- p.37 / Chapter 2.2.1 --- Growth and treatment of soybean seedling --- p.37 / Chapter 2.2.2 --- RNA extraction from root tissue --- p.37 / Chapter 2.2.3 --- RNA denaturing gel electrophoresis --- p.39 / Chapter 2.2.4 --- Generation and testing of single-stranded DIG-labeled PCR probes --- p.39 / Chapter 2.2.5 --- Northern blot analysis --- p.41 / Chapter 2.2.6 --- Transformation of V7/GmCLCl electro-competent Agrobacterium tumefaciens --- p.42 / Chapter 2.2.7 --- PCR screening of transformed Agrobacterium tumefaciens colonies --- p.43 / Chapter 2.2.8 --- DNA gel electrophoresis --- p.43 / Chapter 2.2.9 --- Agrobacterium-mediated transformation of tobacco BY-2 cells --- p.44 / Chapter 2.2.10 --- Verifying the expression of GmCLCl in transgenic tobacco BY-2 cells --- p.45 / Chapter 2.2.11 --- Salt treatment of tobacco BY-2 cells and cell viability assay --- p.46 / Chapter 2.2.12 --- Subcloning of GmCLCl cDNA into pgh21 vector --- p.47 / Chapter 2.2.13 --- In vitro synthesis of GmCLCl cRNA --- p.51 / Chapter 2.2.14 --- Obtaining oocyte from Xenopus laevis ovaries --- p.52 / Chapter 2.2.15 --- Microinjection of GmCLCl cRNA into Xenopus oocyte and oocyte incubation --- p.53 / Chapter 2.2.16 --- Two electrode voltage clamp of Xenopus oocytes --- p.54 / Chapter 3. --- Results --- p.56 / Chapter 3.1 --- Phylogenetic analysis of GmCLCl --- p.56 / Chapter 3.2 --- Expression of GmCLCl in root was induced by NaCl and alkaline condition --- p.60 / Chapter 3.3 --- Construction of GmCLCl transgenic tobacco BY-2 cell line --- p.62 / Chapter 3.4 --- GmCLCl improve NaCl stress tolerance of transgenic tobacco BY-2 cells in a pH dependent manner --- p.67 / Chapter 3.5 --- Subcloning of GmCLCl into pgh21 --- p.70 / Chapter 3.6 --- GmCLCl cRNA synthesis by in vitro transcription --- p.72 / Chapter 3.7 --- Two electrode voltage clamp (TEVC) of GmCLCl cRNA injected Xenopus oocytes --- p.75 / Chapter 4. --- Discussion --- p.81 / Chapter 4.1 --- Implications from phylogenetic and sequence analysis on the function of GmCLCl --- p.81 / Chapter 4.2 --- Electrophysiological characterization of GmCLC 1 by Xenopus oocytes --- p.82 / Chapter 4.3 --- Some plant CLCs contributed in salt tolerance response --- p.84 / Chapter 4.4 --- Relationship between pH and physiological function of plant CLCs --- p.85 / Chapter 5. --- Conclusion and Perspectives --- p.88 / Chapter 6. --- Appendices --- p.90 / Chapter Appendix I: --- Major Chemicals and reagents used in this research --- p.90 / Chapter Appendix II: --- Enzymes used in this research --- p.92 / Chapter Appendix III: --- Major equipment and facilities used in this research --- p.93 / Chapter Appendix IV: --- "Buffer, solution, gel and medium formulation" --- p.94 / Chapter 7. --- References --- p.96

A debilitating illness, pulmonary arterial hypertension (PAH) arises from deleterious remodeling of pulmonary arterioles, leading to increased pulmonary artery pressure, a rise in pulmonary vascular resistance, right sided heart failure and death. The pathogenesis of the disease is incompletely understood; however, certain established pathological features have guided medical treatments to improve mortality rates. For instance, an imbalance of vasoconstrictor molecules, such as endothelin-1, to vasodilator compounds, such as nitric oxide, contributes to excessive pulmonary arterial constriction, and a propensity for pulmonary arterial smooth muscle and endothelial cell proliferation. Therapeutic strategies may aim to restore this imbalance with the use of endothelin receptor antagonists, prostacyclin analogs, and other vasodilating agents. Mutations in the BMPR2 gene, the most common genetic cause of PAH, leads to aberrant TGF-ß signaling, which promotes uncontrollable cell proliferation and pathological changes in pulmonary arterioles. Genetic studies have revealed PAH-associated mutations in several other genes within the TGF-ß signaling pathway. More recently, our research group discovered loss-of-function mutations in the KCNK3 gene encoding the KCNK3 two-pore domain potassium channel in patients with idiopathic and familial PAH. KCNK3 (also referred to as TASK-1, or K2P3.1) represents the first ion channelopathy as a cause of PAH. KCNK3 is expressed in human pulmonary artery smooth muscle and endothelial cells. Loss of KCNK3 channel currents leads to membrane depolarization and predisposes to deleterious pulmonary arterial remodeling. Chapter 1 of my thesis explores the impact of KCNK3 mutations on potassium channel function in cellular models of heterozygous conditions, as all patients with PAH-associated KCNK3 mutations in our study were heterozygous at the KCNK3 gene locus. Furthermore, we explored function of mutant and non-mutant KCNK3 channels in cultured human pulmonary artery smooth muscle cells to better define the electrophysiological consequence of KCNK3 dysfunction, and used a KCNK3-activating pharmacological agent, ONO-RS-082, to gauge the therapeutic potential of KCNK3 as a pharmacological target in PAH. Moreover, the study of KCNK3 channel activity when assembled with the closely related KCNK9 channel provided a platform for exploring the lung-specific phenotype in patients with heterozygous KCNK3 mutations, despite widespread tissue expression KCNK3 in the body. In Chapter 2 of my thesis work, the discovery of a second potassium channelopathy in PAH is characterized. Heterozygous mutations in the ABCC8 gene, encoding the sulfonylurea receptor 1 (SUR1) protein, were found in pediatric and adult patients with idiopathic and familial PAH. SUR1, a beta subunit of the ATP-sensitive potassium channel (KATP), assembles with the pore-forming Kir6.2 alpha subunit to form KATP, a channel sensitive to inhibition by intracellular ATP. At the plasma membrane, KATP inwardly rectifying potassium currents contribute to the resting potential, and may play a pathophysiological role in PAH via dysfunction in pulmonary artery smooth muscle and/or endothelial cells. In this chapter, eight ABCC8 mutations associated with PAH were functionally characterized, and pharmacological agents were employed to examine the therapeutic potential in targeting SUR1-containing KATP channels in PAH. Altogether, the research presented in this dissertation identifies and explores potassium channel dysfunction as a pathogenic mechanism in PAH, due to heterozygous genetic mutations in KCNK3 and ABCC8. Evidence of restoration of mutant KCNK3 and KATP channel function by pharmacological agents suggests that targeting potassium channels as a therapeutic strategy may alleviate the severe morbidity and mortality burden in patients with PAH.

The pedigree of voltage-gated sodium channels spans the millennia from eukaryotic members that initiate the action potential firing in excitable tissues to primordial ancestors that act as enviro-protective complexes in bacterial extremophiles. Eukaryotic sodium channels (eNavs) are central to electrical signaling throughout the cardiovascular and nervous systems in animals and are established clinical targets for the therapeutic management of epilepsy, cardiac arrhythmia and painful syndromes as they are inhibited by local anesthetic compounds. Alternatively, bacterial voltage-gated sodium channels (bNavs) likely regulate the survival response against extreme pH conditions, electrophiles and hypo-osmotic shock and may represent a founder of the voltage-gated cation channel family. Despite apparent differences between eNav and bNav channel physiology, gating and gene structure, the discovery that bNavs are amenable to crystallographic study opens the door for the possibility of structure-guided rational design of the next generation of therapeutics that target eNavs. Here I summarize the gating behavior of a bacterial channel NaChBac and discuss mechanisms of local anesthetic inhibition in light of the growing number of bNav structures. Also, an interesting novel observation on cross-lineage modulation of NaChBac by eNav beta subunit is reported. This auxiliary subunit modulation is isoform specific and I show the discrete effects of each isoforms on NaChBac, with functional and biochemical analysis. I also report a novel mutation that alters inactivation kinetic drastically and a possible mechanism of NaChBac inactivation is discussed.

The secretion of the acqueous component of tears is dependent on the uneven distribution and nature of ion channels across lacrimal gland acinar cell membranes. Models to describe such secretion in lacrimal glands are based primarily on one species, the rat and have been developed in conjunction with observations of channel function and secretion from salivary and pancreatic exocrine glands, which are structurally similar to lacrimal glands. The applicability of these models to all species is unknown and furthermore none takes into account volume regulation mechanisms in these acinar cells. Therefore, the aim of this thesis was to broaden the basis for such models by identifying the major membrane ionic channels, characterising their mode of activation and determining their possible physiological roles in rabbit superior lacrimal gland acinar cells. Initially, a culture system for maintaining acinar cells for short and long terms was developed. This allowed for electrophysiological techniques to be employed to study the role of ionic channel activity in the secretion of the aqueous component of tears and cell volume regulation. This study demonstrates a number of unique findings and indicates that the models currently used to describe lacrimal gland secretion are not applicable to all species and need modifications which take into account cell volume regulation / Doctor of Philosophy (PhD)

No description available.

ClC proteins are chloride channels and transporters that are found in a wide variety of prokaryotic and eukaryotic cell-types. The mammalian chloride channel ClC-1 is an important modulator of the electrical excitability of skeletal muscle. The Torpedo electric-organ chloride channel, ClC-0 is structurally and functionally similar to ClC- 1. These proteins are referred to as the muscle-type ClC channels. The present work identifies several functional differences between the muscle type channels, and explores the structural basis of these and other previously reported differences. First the temperature dependence of ClC-1 channels was quantified. These calculations revealed distinct contrasts to previously published measurements of ClC-0 temperature sensitivity, indicating differences between the channels in the structural rearrangements associated with channel gating. Next the effect of extracellular ion substitution on ClC-0 function was examined. These measurements suggested that occupancy of an anion binding-site on the extracellular side of the selectivity-filter stabilises the open state of the channel, and that the diameter of the channel pore increases during channel opening. Three-dimensional models of the muscle-type channels were constructed based on the atomic coordinates of prokaryotic homologues. Differences in selectivity between ClC-0 and ClC-1 could be rationalised, in part, by differences in the chemistry of the narrow constriction of the channel pore. The major structural divergence between the muscle-type channels occurs in the expansive intracellular carboxy terminus. Replacing this region of ClC-1 with the corresponding region from ClC-0 resulted in distinct changes in common gating of the channel. These experiments rigorously characterise the dependence of ClC-1 function on temperature and the effect of foreign anionic-substrates on ClC-0 function. The results identify important residues involved in ionic selectivity of the channels, and validate the use of high-resolution prokaryotic channel structures as a predictive tool for studying the muscle-type channels. They also demonstrate that the carboxy-terminal of the channels is an important determinant of common gating. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Bibliography: leaves 97-112. / vii, 112 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 2003

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: Michael J. O'Donnell. Includes bibliographical references (leaves 279-310).

Neuronal nicotinic acetylcholine receptors can have their function modulated by zinc. Depending on concentration and subunit composition, zinc either inhibits or potentiates receptor function. The zinc ion potentiates the alpha4beta4 receptor at non-agonist binding interfaces or "pseudo sites." Zinc potentiation is reduced if certain residues are mutated or spatially interfered with. The residue contributing most to this potentiation reduction effect is histidine 162 on the alpha4 subunit. The anthelmintic compound levamisole potentiates acetylcholine response of certain neuronal nicotinic receptors. Levamisole and its functional analogues morantel, oxantel, and pyrantel all were found to potentiate alpha4beta4 receptors at low (µM) concentrations and inhibit them at high (mM) concentrations. Oxantel showed the greatest degree of potentiation, about a third of the maximal zinc potentiation measured. Oxantel was screened using the substituted cysteine accessibility method (SCAM) against the residue histidine 162 as well as nearby alpha4 residues histidine 61 and glutamate 59 and the beta4 residue aspartate 195. Screening was carried out by mutating said residues into cysteine, followed by covalent linkage with a disulfide bridge of that residue with a methanethiosulfonate compound. SCAM experiments allowed testing of the effects of a single residue and the area immediately adjacent to it. Receptors that lost zinc potentiation capacity from site-directed mutagenesis at the his 162 residue and subsequent methanethiosulfonate reaction still showed regular potentiation following oxantel treatment. Although these compounds exhibit similar biphasic potentiation dose-response curves as zinc, their mechanism for potentiation is not through the same mechanism.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 121-137).