Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct. 30, 2007). Includes bibliographical references (p. 165-174).

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2013 / The chloride intracellular channel CLIC1 is an anion channel protein that has been implicated in a number of physiological processes. It is fascinating in that it is synthesised as a soluble monomer that is able to reversibly bind membranes without the aid of a membrane-targeting tag or receptor. CLIC1 membrane binding is promoted by low pH and involves separation of the N- and C-domains and subsequent refolding of the N-domain, which traverses the membrane as an α-helix. At the low pH of a membrane surface, pH 5.5, soluble CLIC1 demonstrates decreased conformational stability and forms a partially unfolded intermediate state under mild denaturing conditions. In this study, these pH-effects are proposed to occur as a result of low pH-induced protonation of two conserved glutamate residues, Glu85 and Glu228. Both are involved in domain-maintaining interactions and are proposed to form part of an electrostatic network of pH-sensitive residues. At low pH, protonation of these glutamates would break their electrostatic interactions, allowing separation of the domains. To investigate this possibility, Glu228 and Glu85 were mutated to leucine residues. Each variant protein was then investigated at pH 7.0 and pH 5.5 and results were compared to the wild-type. Secondary and tertiary structures were examined using far-UV circular dichroism and fluorescence spectroscopy, respectively. Conformational flexibility was investigated with limited thermolysin proteolysis. Stability was studied using thermal and urea-induced equilibrium unfolding. The unfolding intermediate state was detected using ANS binding and its structure was characterised. While neither residue substitution caused global structural perturbations, both destabilised the structure and promoted intermediate formation at pH 5.5. This was particularly evident for the E85L variant, which also formed a significant intermediate population at pH 7.0. It was concluded that the interactions of Glu228 and Glu85 are involved in maintaining the CLIC1 native state. Additionally, the lack of pH-dependence of intermediate formation in the E85L variant suggested that Glu85 is likely to function as a pH-sensor. It is thus involved in the „priming‟ of the CLIC1 structure for the conformational changes that may lead to membrane binding.

No description available.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves: 152-155). Also available on the Internet.

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

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

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 77-87).

Lactic acid, which induces pH depression, leads to ATP efflux from muscle to extracellular space: it was reported that CFTR was involved in this process. However, the mechanism by which lactic acid activated CFTR and brought about the ATP release is still unknown. This study was performed to investigate (1) what channels may be involved or even conduct ATP release, and (2) how lactic acid activated CFTR. Expression of the possible channels that may conduct ATP release in L6 cells was investigated using RT-PCR: ClC-2, ClC-3, ClC-7, CACC, VDAC, connexin 40, connexin 43 and pannexin 3 were expressed in L6. Incubation of cultured L6 cells with lactic acid (10 mM) increased the extracellular ATP from 0.6 ± 0.06 to 1.1 ± 0.09 nM (P ? 0.05), indicating that lactic acid stimulated ATP efflux in vitro. The non-specific chloride channel inhibitor, DIDS, failed to abolish the lactic-acid-induced ATP release, suggesting that DIDS-sensitive chloride channels were not involved in the ATP efflux. Among the non-specific inhibitors of connexin channels, gadolinium inhibited acidosis-induced ATP efflux, but carbenoxolone failed to inhibit it, and so the role of connexins remains uncertain. The specific inhibitor of CFTR, CFTRinh-172, and the non-specific open-channel blocker of CFTR, glibenclamide, both abolished the acidosis-induced ATP release, but another specific inhibitor of CFTR, GlyH-101, which blocks CFTR from the external side, failed to abolish the ATP release, suggesting that acidosis-induced ATP is dependent on CFTR-activation, but does not involve ATP moving through the CFTR chloride channel. We hypothesize that, at low pH, the Na+/H+ exchanger (NHX) extruded H+ out of the cell and the resulting intracellular Na+ was transported out by Ca2+/Na+ exchanger (NCX); the localized increase in Ca2+ activated adenyl cyclase (AC), thus elevating intracellular cAMP; cAMP-activated-PKA then phosphorylated CFTR, which regulated an ATP release channel. KT-5720, an inhibitor of PKA, abolished the acidosis-induced ATP release, and forskolin, an agent that elevates cAMP, stimulated it, suggesting that the cAMP/PKA pathway was involved. The specific inhibitor of NCX, SN-6 and KB-R7943, both abolished the acidosis-induced ATP release, supporting a role for NCX in mediating this process. However, amiloride, the non-specific inhibitor of NHX failed to abolish ATP efflux. The whole cell Cl- currents were studied in L6 cells: lactic acid increased the whole cell currents from 2.33 ± 0.10 to 3.54 ± 0.34 nA (P ? 0.05), and this lactic-acid-induced increase in Cl- current could be inhibited by CFTRinh-172, suggesting that the CFTR Cl- channel was opened at low pH. Moreover, forskolin increased whole cell Cl- currents, which supported a role for the cAMP/PKA pathway in the lactic-acid-induced increase in CFTR current. These data confirm that CFTR is involved in the lactic-acid-induced ATP release from L6 cells. The roles of the NCX and cAMP/PKA pathway in activating CFTR at low pH are supported, but further studies are required to determine whether the NHX is involved in CFTR activation and whether connexins participate in ATP release. / published_or_final_version / Physiology / Master / Master of Philosophy

Contracting skeletal muscle releases ATP into the interstitial space where it is subsequently broken down to adenosine by the action of ecto-5’-nucleotidase. Both ATP and adenosine are vasodilators that contribute to the exercise hyperaemia. However, the mechanism for the release of ATP from muscle during exercise remains unknown. Cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from muscle at low intracellular pH: this study was performed to investigate whether CFTR was involved in the ATP release from skeletal muscle during contractions. Experiments were performed in rats anaesthetised with sodium pentobarbitone and breathing spontaneously. A microdialysis probe was placed in one gastrocnemius muscle: ATP was determined in interstitial microdialysate samples using a bioluminescence assay. The sciatic nerve was stimulated to induce two bouts of muscle contractions, separated by a recovery period of 40 mins; one of the inhibitors was administered prior to the second bout of contractions. Muscle contractions elevated the interstitial ATP by 1500 to 3000%. In the control experiments, no drug was given: both the contractile force and the increase in interstitial ATP were reproducible in repeated contraction bouts. Infusion of a specific inhibitor of CFTR, CFTRinh-172, did not alter the contractile force, but significantly lowered the interstitial ATP during muscle contractions, suggesting that CFTR was involved in the contraction-induced ATP release. Similarly, infusion of the Protein Kinase A inhibitor, KT5720, significantly reduced interstitial ATP during muscle contractions without altering contractile force, suggesting that CFTR in skeletal muscle is activated through the cAMP/PKA pathway. The increase in interstitial ATP during muscle contraction was also inhibited by the Na/H exchanger inhibitor, amiloride, or the Na/Ca exchanger inhibitor, SN6. It has been also shown that two gap junction hemichannel inhibitors, gadolinium and carbenoxolone, could attenuate the increase of ATP during muscle contraction. These data suggest that CFTR, activated through the cAMP/protein kinase A pathway, is involved in the ATP release during muscle contraction, and that activation of the Na/H exchanger and Na/Ca exchanger was also required, indicating that the signal transduction mechanism for CFTR activation during muscle contractions may be similar to that which is reported to occur at low pH. The preliminary data showed that the gap junction hemichannels might mediate the ATP release from skeletal muscle cells during muscle contraction. / published_or_final_version / Physiology / Master / Master of Philosophy