The glutamate-gated chloride (GluCl) channel family of receptors are members of the ligand-gated ion channel (LGIC) family. In addition to being essential for physiological and behavioural aspects of an organism, they are also exploited as a target for the drug ivermectin. A novel GluCl subunit in C. elegans, GLC-4, was characterized in order to further understand the diversity of GluCls and their implications in biological and behavioural processes. Sequencing of cDNAs generated by RT-PCR indicate that GLC-4 possesses the typical features of a GluCl subunit. In addition, they also suggest possible alternative splicing of glc-4 resulting in a slightly truncated transcript. A glc-4 mutant, glc-4(ok212), was used to investigate the role of GLC-4 in C. elegans ivermectin sensitivity and behaviour. Glc-4(ok212) worms were found to be hypersensitive to low concentrations of ivermectin and experienced a slight hyperreversal behaviour. Genetic, molecular, and electrophysiological evidence is also provided suggesting an interaction between GLC-4 and AVR-14, another GluCl subunit in C. elegans. We hypothesize a direct association between GLC-4 and AVR-14 which together form a heteromeric channel in vivo .

ClC proteins encompass a large protein family consisting of both voltage-dependent chloride channels and chloride/proton exchangers that are found in both eukaryotes and prokaryotes. These proteins mediate Cl- flux across the plasma membrane or intracellular membranes of many cell types including neurons, epithelial cells, and skeletal muscle in mammals. Mutations in genes encoding these channels also contribute to several human diseases. The mechanism of ion conduction through ClC proteins is becoming better defined, largely due to the availability of a crystal structure of a bacterial ClC transporter. Because crystal structures only capture a snapshot a protein in a single conformation, however, the large conformational changes associated with channel opening and closing have remained largely undefined. In the cation channel field, ion conduction and conformational changes that occur during channel gating have been studied using peptide toxin inhibitors isolated from animal venoms. However, only one peptide toxin inhibitor of a chloride channel of known molecular identity has ever been identified. Georgia anion toxin 1 (GaTx1), inhibits the CFTR chloride channel, which is unrelated to ClC proteins on the levels of both three dimensional structure and primary sequence. Here, we describe the characterization of the inhibitory activity of Leiurus quinquestriatus hebraeus scorpion venom against the ClC-2 chloride channel. We found that the venom from this scorpion contains a peptide component that is capable of inhibiting the ClC-2 chloride channel. This component was isolated using standard chromatography techniques, and found that the active component is a 3.2 kDa peptide composed of 29 amino acids. We showed that the active toxin, Georgia anion toxin 2 (GaTx2), interacts with ClC-2 with an affinity in the picomolar range, and appears to slow channel opening. Finally, GaTx2 is not capable of inhibiting other members of the ClC protein family, other major chloride channels, or voltage-gated potassium channels. This toxin will provide a new tool for structure/function studies of ClC-2, and will hopefully serve as only the first toxin inhibitor available for this protein family.

Thesis (Ph. D.)--University of Nevada, Reno, 2007. / "December, 2007." Includes bibliographical references (leaves 214-241). Online version available on the World Wide Web.

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

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 10, 2007) Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Nevada, Reno, 2008. / "May, 2008." Includes bibliographical references. Online version available on the World Wide Web.

Bestrophins are a family of calcium (Ca²⁺) -activated chloride (Cl⁻) channels (CaCCs) with functional importance in eye physiology. Mutations to the VMD2 gene, which encodes the Best1 protein, cause an array of degenerative eye disorders called bestrophinopathies, which result from aberrant CaCC activity of the Best1 channel in the pigmented epithelium of the retina. While there are four bestrophin paralogs in mammals (Best1-4), the only current structures are of Best1 homologs. The structure of the prokaryotic homolog of Best1 from Klebsiella pneumonia (KpBest) was previously solved in this lab, representing the first structure of a Best1 homolog at the time. This initial study laid the foundational groundwork in the field and contributed significant knowledge to understanding the bestrophin structure-function relationship. Nevertheless, significant questions remain regarding bestrophin function, such as the molecular determinants underlying its Ca²⁺-dependent gating and anion selectivity. This dissertation uses single-particle cryogenic electron microscopy paired with electrophysiology to probe the structure-function relationship of mammalian bestrophins under different buffer conditions and reveals conformational dynamics involved in gating of wild-type channels. Key regions of the channel contributing to its function are described at the atomic level leading to development of a gating model to explain Ca²⁺-dependent activation and inactivation in mammalian bestrophins.

No description available.

No description available.

No description available.