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 121-137).

Karenia brevis is the major harmful algal bloom-forming species in the Gulf of Mexico, and produces neurotoxins, known as brevetoxins, that cause large fish kills, neurotoxic shellfish poisoning, and human respiratory distress. Brevetoxins are polyethers that bind voltage-sensitive sodium channels, opening them for prolonged periods of time. Clonal cultures of K. brevis exhibit unique brevetoxin profiles, which not only differ from one another, but also change when subjected to different environmental conditions. The brevetoxin structures were elucidated 30 years ago without any breakthroughs for the biosynthetic pathway. These unique ladder-like polyethers have 10 (PbTx-1) or 11 (PbTx-2) rings, indicating that they are synthesized as secondary metabolites by polyketide synthases. The extensive size of the genome and the lack of histones and nucleosomes combined with the additional regulatory step of a trans-splicing spliced leader sequence make normal molecular techniques ineffective in determining the genes involved in toxin synthesis. The goal of this project is to identify a potential link between toxin, gene, and function. One objective is to take the next step towards identifying the genes associated with the synthesis and regulation of brevetoxins and to help elucidate the hypothesized gene clusters of multi-protein enzymatic complexes involved in brevetoxin production, one for each backbone. The second objective is to make an effort to determine the in vivo function of the costly brevetoxins by identifying possible ion channels, which could be osmotically regulated by the toxins. Genes for polyketide synthases (PKS) were identified in K. brevis, obtained from Expressed Sequence Tag (EST) libraries. In this work, reverse transcription polymerase chain reactions (RT-PCR) were used to generate pools of complementary DNA (cDNA), which was used in real-time quantitative polymerase chain reactions (qPCR) to give relative amounts of PKS transcripts. K. brevis clones have shown a significant increase in toxin production after a rapid shift from high salinity to low salinity, indicating a regulation of brevetoxin synthesis. To gain a better understanding of regulation of toxin production during algal blooms, we compared the toxin levels under different conditions to the transcript levels of PKS genes, as determined by quantitative RT-PCR. In a separate line of investigation, an in silico analysis of the EST library was performed to identify ion channel genes expressed by K. brevis, which may be the in vivo binding site of brevetoxin. The information generated from this project will help to elucidate the effects of environmental variations on toxin production and the biological function of toxin production -- valuable information for the shellfish industries and public health.

Potassium ion channels play a key role in the generation and propagation of action potentials. The S4-S5 linker peptide (L45) is believed to be responsible for the anesthetic/alcohol response of voltage-gated K+ channels. We investigated this region to define the structural basis of 1-alkanol binding site in dShaw2 K+ channel. L45 peptides derived from dShaw2 and hKv3.4 K+ channel, which, if part of the complete channel, demonstrate different sensitivity to 1-alcohols. Specifically, dShaw2 is alcohol sensitive and hKv3.4 is alcohol resistant. Structural analysis of L45 with NMR and CD suggested a direct correlation between alpha-helicity and the inhibition of dShaw2 channel by 1-butanol. We used CD and NMR to determine the structure of L45 peptides in micelles and vesicles. We measured spin-lattice relaxation time (T1) and determined the location and surface accessibility of L45 in micelles. These experiments confirm that L45 of dShaw2 adopts an α-helical conformation, partially buried in the membrane and parallel to the surface. The binding and accumulation of rev proteins to an internal loop of RRE (rev responsive element) of unspliced mRNA precursors is a key step of propagation of human immunodeficiency (HIV) virus. Molecules that interfere with this process can be expected to show anti-HIV activity. Our work is based on an assumption that zinc fingers could compete with rev proteins, therefore impeding the life cycle of HIV and stopping its infection. We studied the influence of different cations, anions, and the concentration of salts and osmolytes on the binding affinity with Polyacrylamide Gel Electrophoresis (PAGE) and Isothermal Titration Calorimetry (ITC). We conclude that the types of anions and/or cations and their concentrations affect the enthalpy and entropy of the binding interacitons. Using a gel assay, we confirm that there are three products in RNA-Protein reaction, and both EDTA and salts (and their concentrations) in the gel or samples interfere with RNA-protein complex mobility.

This dissertation will present the four manuscripts I published or am ready to publish on the study of the pathophysiology of migraine headache. The first chapter will discuss the background of the current understanding of migraine pathophysiology. Chapter 2 is focused on studying how Transient receptor potential vanilloid 4 (TRPV4) might play a role in migraine headache. Chapter 3 is the study of a novel cell type: dural fibroblasts might also play an active role in migraine headache. Chapter 4 is discussing Norepinephrine's role in headache pathophysiology. Chapter 5 is studying the combined effect of Acid and ATP in the pathophysiology of migraine headache. The dissertation will end in a conclusion in Chapter 6.

Nicotinic acetylcholine receptors (nAChR) are neurotransmitter-gated ion channels that exist as a family of subtypes defined by unique subunit compositions. nAChR containing α6 subunits (α6*-nAChR) have attracted interest because α6 subunits are thought to be localized in brain regions implicated in reward, mood and drug dependence. To provide new information necessary toward a more complete understanding of roles of α6*-nAChR in neuropsychiatric health and disease, three lines of investigation were pursued. A set of stably transfected, human, immortalized cell lines were generated that heterologously express nAChR α6 subunits in combination with other nAChR subunits found in reward brain regions (nAChR subunit combinations α6β2, α6β4, α6β2β3, α6β4β3, α6β2β3α5, α6β4β3α5, α6α4β2β3 and α6α4β4β3). The α6α4β2β3 combination may have a functional response to epibatidine that differs from that of the α4β2 nAChR. A unique binding site was identified in cells transfected with the α6β4β3α5 nAChR subunit combination. Messenger RNA fluorescence in situ hybridization (mRNA FISH) studies established regional and celluar distribution of nAChR α6 subunit mRNA in the mouse brain. The third line of study extended this work to examine potential co-expression of nAChR α6 subunits and glutamic acid decarboxylase (GAD) or tyrosine hydroxylase (TH) as labels of GABAergic and dopaminergic/catecholaminergic neurons respectively, using tandem mRNA FISH and fluorescence immunohistochemistry. nAChR α6 subunit signal in the substantia nigra (SN) and ventral tegmental area (VTA) was congruent with previous studies. Message was also detected in the amydala, dentate gyrus, striatum, zona incerta, and cingulate, entorhinal, perirhinal, piriform, and prelimbic cortices. nAChR α6 mRNA was coexpressed with GAD in the amygdala, dentate gyrus, striatum, SN, VTA and cingulate, entorhinal, prelimbic and prelimbic cortices. TH was exclusively co-localized with nAChR α6 mRNA in the SN and VTA. Findings suggest extended roles for α6*-nAChR in the brain, particularly in the control of GABAergic neuronal activity and/or GABA release. These studies provide new insights into the composition of α6*-nAChR, the localization and cellular origins of nAChR α6 subunit expression. Data collected suggest roles for α6*-nAChR in many brain regions, including those involved in higher order processes involved in drug dependence and reward, and in modulation of inhibitory neurotransmission.

Evidence is presented that Lymnaea contains homologues for mammalian Cav3 and NALCN 4-domain ion channels, which retain key amino acid sequence motifs that differentiate these channels from other 4-domain types. Molecular cloning and heterologous expression of the first invertebrate Cav3 channel cDNA from Lymnaea confirms that it indeed is a true homologue to mammalian Cav3 channels, retaining some hallmark biophysical and pharmacological features1. Interestingly, the Lymnaea Cav3 channel gene also exhibits alternative splicing that is conserved with mammalian Cav3.1 and Cav3.2 channels, with homologous exons 8b in the I-II linker (Cav3.1) and 25c in the III-IV linker (Cav3.1 and Cav3.2), that can selectively be included or omitted from the full length channel. We show that the developmental and spatial expression patterns of these splice variants are remarkably conserved, and that these splice variants produce analogous changes in membrane localization and biophysical properties when channels are expressed in HEK-293T cells. The Lymnaea Cav3 channel gene also undergoes alternative splicing in the domain II P-loop, with mutually exclusive exons 12A and 12B that code for a large portion of the P-loop just upstream of the selectivity filter. Such splicing is a novel discovery that is not conserved with vertebrates or any other deuterostome animal, all of which only contain 12A homologues of exon 12. However, protostome animals including Lymnaea stagnalis, Drosophila melanogaster, and C. elegans all have mutually exclusive 12A and 12B exons in their Cav3 channel genes. Evidence is presented that exon 12A is likely the ancestral exon for the domain II P-loop, and that alternate exon 12B evolved later. Furthermore, although the two Lymnaea variants possess the same selectivity filter motifs characteristic for Cav3 channels (i.e. EEDD), they exhibit dramatic differences in calcium vs. sodium selectivity, without significant differences in biophysical properties. This is the first account of alternative splicing used to modulate ion selectivity in a Cav3 channel homologue, and given that calcium is such an important electrogenic signaling molecule, these alterations are expected to have profound physiological implications. Amazingly, Lymnaea NALCN was also found to undergo alternative splicing in the domain II P-loop, but in this case, the entire P-loop is replaced by mutually exclusive exons 15a and 15b such that the selectivity filter is converted from the proposed non-selective sodium-permeable configuration (15b/EKEE; EEKE in mammals, nematodes and insects), to a calcium channel-like pore (15a/EEEE). Thorough phylogenetic analysis reveals that NALCN is extremely unconventional, in that alternative splicing has frequently and independently evolved to alter the selectivity filter in domains II or III, in multiple animal clades. Furthermore, the ancestral NALCN channel most likely contained an EEEE pore. This work brings into question NALCN???s proposed role as a major leak sodium conductance that depolarizes neurons to help set the resting membrane potential, since some species possess only an EEEE variant, and based on homology to other 4-domain ion channels, this should render the channel calcium-selective. Unfortunately, heterologous expression and electrophysiological characterization of the two Lymnaea NALCN isoforms was unsuccessful, corroborating with others the inability to record NALCN ionic currents in heterologous systems.

This thesis describes the development of an ion channel system exhibiting dissipative assembly characteristics. In this system an active transporter based on an oligoester fragment terminated in a thioester of 6-aminohexanoic acid (HO2C-Hex-Adip-OctS-Hex-NH2) undergoes thioester cleavage to form a thiol terminated oligoester (HO2C-Hex-ADip-Oct-SH). This fragment was expected to be inactive for ion transport but previous work showed high activity in planar bilayer experiments. In this work, the high activity was shown to be due to the oxidized form of the thiol, the disulfide HO2C-ADip-Oct-SS-Oct-ADip-Hex-CO2H. Air oxidation was found to be quite rapid for the thiol based on ESI-MS (negative ion) and HPLC analysis. Under anaerobic conditions, the thiol fragment was an inactive species for ion transport. In situ air oxidation initiated transport activity associated with the disulfide. The transporter HO2C-Hex-Adip-Oct-Hex-NH2 was active in planar bilayer experiments and was compared to the disulfide via activity grids. The activity of these two compounds was shown to be distinct from each other by conductance and channel duration differences. The activity of HO2C-Hex-Adip-Oct-Hex-NH2 was shown to die off in a period of 30 minutes at pH 8.2. Techniques were developed to stimulate and monitor activity and bilayer quality so that an inactive condition could be confirmed. The addition of Pr-S-Hex-NH3+-Cl as a fuel was shown to extend the activity of HO2C-Hex-Adip-Oct-Hex-NH2 by eight-fold in 1M CsCl electrolyte. Previous work had established the capability of thioester exchange reactions by a reaction between Pr-S-Hex-NH3+-Cl and benzyl thiol in a homogenous solution. The extended activity indicated that the same process may occur in a heterogeneous bilayer system. An inactive system created by the die-off in activity of HO2C-Hex-Adip-Oct-S-Hex-NH2 was treated with Pr-S-Hex-NH3+-Cl to regenerate activity. This cycle could be repeated once the activity died off again. All these findings are consistent with the dissipative assembly of a membrane transport system. / Graduate / 0490

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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.