In living systems, ion channels are membrane transport proteins that provide pathways for the passive diffusion of ions through lipid membranes. The flow of ions across membranes is the basis of many important physiological processes, including but not limited to the regulation of membrane potential, transepithelial transport and cell volume. While many efforts have been made to understand the biological roles of natural ion channels, the biological activities of artificial ion channels remain largely unknown. Recently, it was reported that a small molecule 1, which forms synthetic chloride (Cl–) channels in membranes via self-assembly, is capable of modulating vascular functions. In this thesis, novel small molecules that are structurally similar to 1 are shown to form artificial ion channels in membranes. Together with 1, the effects of these small molecules on the contractile activities of smooth muscles and epithelial ion transport are explored. The therapeutic implications of the findings are also discussed. A collection of small molecules was screened using liposome-based fluorescence assays. In these assays, the ability of the synthetic compounds to modulate membrane potential was monitored. The screening yielded compound 3 that formed synthetic potassium (K+) channels in liposomal membranes, although the liposome-based fluorescence experiments suggested that 3 also transported Cl–. Two derivatives of 3, namely, compounds 2 and 4 were also examined. Single-channel recording experiments suggested that 2 forms synthetic Cl– channels in liposomal membranes. The effects of compounds 2 and 3 on the functions of the vascular smooth muscle are explored. Using confocal imaging, it was shown that both 2 and 3 counteracted the effects of high-K+ depolarizing solution on membrane potential and intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells. 2 and 3 also relaxed mice aortic rings pre-contracted with high-K+ solution. These observations can be explained in terms of the Cl– transporting functions of 2 and 3. To determine the potential for developing the compounds into bronchodilators, the effects of compounds 1 and 3 on the contractile activities of the airway smooth muscle (ASM) were explored using organ bath technique. The contractile activities of the trachea isolated from Sprague-Dawley (SD) rats were first characterized. Among the contractile agents used, only potassium chloride (KCl), cholinergic agonists, serotonin and endothelin-1 were contractile to the SD rat trachea. 1 and 3 relaxed the ASM pre-contracted with KCl, whereas the contractions induced by other agonists were not affected. The ability of compounds 2, 3 and 4 to modulate ion transport across cultured epithelia was tested by the short-circuit current measurement technique. It was shown that the compounds were capable of inducing Cl– secretion when applied to the apical side of airway and colonic epithelia. Importantly, the synthetic compounds induced apical Cl– secretion in immortalized cystic fibrosis (CF) bronchial epithelia. This suggests that the synthetic compounds may be used to correct the anion transport defect in CF epithelia. In summary, the small-molecule based synthetic ion channels demonstrated two important general functions of natural ion channels, namely, the regulation of membrane potential and epithelial ion transport. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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The mechanism of zinc transport has been investigated in red cells from normal humans, lampreys, sheep, sickle cell anaemia patients and in bovine chondrocytes. In all the cell types investigated except for lamprey red cells, zinc transport is mainly via the anion exchanger (band 3), which accounts for over 80% of total measured zinc uptake, when the medium contains no zinc binding ligands. Zinc uptake via the band 3 pathway is stimulated by the presence of bicarbonate (5mM) and inhibited by treatment with DIDS or SITS (10andmu;M). This anion-dependent mechanism represents the major route for zinc transport across the cell membrane in vitro. The presence of the zinc binding ligands albumin and histidine in the media greatly reduced the uptake of zinc via the anion exchanger due to the decrease in free zinc concentration. Histidine, in addition to its chelating effect, shows a specific facilitating effect on zinc uptake in all the cell types. This stimulating effect of histidine was stereospecific (significantly different between L-, and D-histidine) in red cells from normal humans and sickle cell anaemia patients, but not in red cells from lampreys, sheep, and bovine chondrocytes. Evidence from all cell types strongly suggests that the stimulus is due to the cotransport of zinc and histidine via the histidine transport systems, which are system L, and y* in normal human and sickle red cells; a non-stereospecific L-like system in lamprey red cells and bovine chondrocytes; system C or unknown specific histidine transporter in sheep red cells. The amino acid linked zinc uptake may represent a physiologically significant mechanism for zinc transport into cells.

Electrolyte transport in the intestine of the common brushtail possum (Trichosurus vulpecula) differs from that observed in eutherian mammals. This study has used molecular physiology to identify and characterise the expression and distribution of membrane transporters potentially responsible for these differences in electrolyte transport in the possum intestine. In the possum ileum, secretagogues stimulate an electrogenic Cl⁻-independent HCO₃⁻ secretory response but secretagogue-stimulated Cl⁻ secretion does not occur in this tissue. Based on the ion dependence and pharmacology of the stimulated secretory response, the expression of the cystic fibrosis transmembrane conductance regulator (CFTR), pancreatic Na⁺ HCO₃⁻ cotransporter (pNBC) and Na⁺ K⁺ 2Cl⁻ cotransporter (NKCC1) were investigated in the ileum. Reverse transcription PCR experiments showed that CFTR, pNBC and NKCC1 mRNA transcripts were expressed in the ileal epithelium. It was then demonstrated by in situ hybridisation that both CFTR and pNBC were localised predominantly in the crypts and the levels of expression decreased along the crypt-villous axis towards the lumen. Significantly, the in situ hybridisation results showed that there were low levels of NKCC1 transcript in the ileal epithelium. Western blot studies confirmed that mature CFTR and pNBC proteins were expressed in the ileum, while NKCC1 protein was not detected. The findings of the present study suggest that the absence of Cl⁻ secretion in the ileum is because NKCC1 expression is not elevated in the epithelium. The expression of mature CFTR and pNBC protein suggest that these membrane transporters are involved in the stimulated electrogenic HCO₃⁻ secretory response. The evidence also suggests that CFTR may mediate HCO₃⁻ efflux in the ileum. In contrast, secretagogues do not stimulate an electrogenic secretory response in the proximal and distal colon. This study has shown that CFTR, NKCC1 and pNBC proteins are expressed in the proximal and distal colon. Both NKCC1 and pNBC transcripts were localised to the crypt base in the proximal colon. However, it was shown that CFTR has a punctate distribution and the transcript was predominantly observed in the upper crypt and surface cell region. This study indicated that NKCC1 and pNBC were distributed in a different region of the epithelium compared to CFTR. It was concluded that the distribution of these membrane transporters in different regions of the epithelium accounts for the absence of a stimulated electrogenic secretory response in the possum colon. Given that no stimulated electrogenic secretory response is observed in the colon, it is suggested that HCO₃⁻ secretion by the ileum may have an important physiological role in maintaining an appropriate fluid and pH composition for fermentation in the colonic lumen.

142 leaves : graphs ; 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 Mathematical Physics, 1980

Thesis (MSc.(Hons)) -- University of Western Sydney, 2002. / "A thesis submitted as fulfilment of the requirements for the Degree of Master of Science ( Honours)'' Bibliography: leaves 171-179.

Thesis (M.S. Ed.)--Baylor University, 2008. / Includes bibliographical references (p. 73-77)

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / "May 1997." Typescript. Vita. Includes bibliographical references (leaves 212-236). Also available on the Internet.