Thesis (M.S.)--University of Wisconsin--Madison, 1943. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaf 19).

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Neurosciences. Includes bibliographical references. Available online via OhioLINK's ETD Center.

The nicotinic cholinergic receptor protein of the fish electric organ, Torpedo fuscomaculata, has been isolated, purified and shown to represent a true model for the nAChR from other species and higher vertebrates. It is an integral membrane protein composed of four different subunits, tightly associated with other functional, but non-specific proteins. Purification of the nicotinic cholinergic receptor by chromatofocusing demonstrates an improved method over that of affinity and ion-exchange chromatography. Gel chromatography and SDS-polyacrylamide gel electrophoresis show evidence of four subunits; a(40-44 kDa), 6(53 kDa ),'Y(63 kDa) and 6(66 kDa) despite some degradation of receptor molecules by intracellular proteases. Spectrophotometric and fluorimetric studies of receptor-ligand interactions, show the functional and chemical integrity of the receptor to remain intact after solubilisation. The effect of cholinergic ligands on purified receptor preparations indicate quenching of the intrinsic fluorescence of the receptor. Agonists, like acetylcholine, bind and cause local conformational transitions, changing the active region from a hydrophobic to a hydrophilic environment. This phenomenon is illustrated by the 10-fold increase in fluorescence when the receptor is in a desensitised state. Antagonists, such as d-Tubocurarine, block this conformational transition. In vitro rectus abdominis muscle preparations . show the nitrosamines, dimethylnitrosamine and diphenylnitrosamine, to be true agonists of the nAChR. However their low affinity and specificity for the receptor precludes them as photoaffmity labelling agents. Photoactivation of dimethylnitrosamine occurs when associated with an acidic hydrogen at the active site of the receptor, suggesting energy-transfer labelling to be more facile than photoaffmity labelling. The membrane-bound receptor, in the presence of these nitrosamines, undergoes conformational transitions regulating the opening and closing of the ion-channel. Desensitisation and receptor activation are shown to involve one and the same molecular transition.

The nicotinic acetylcholine receptor (nAChR) is a pentameric ligand-gated ion channel (pLGIC) and is a core component of the neuromuscular junction, facilitating fast synaptic transmission leading to muscle contraction. Mutations to the human adult muscle nAChR lead to various forms of congenital myasthenic syndrome (CMS), a disease characterized by progressive fatigable muscle weakness. A central channel pore constriction formed by a ring of five leucine residues (L9’) forms part of the nAChR channel gate. CMS-causing mutations in the L9’ residues lead to a form of CMS that results in longer channel opening times and a delayed signal decay. To understand better how L9’ mutations in the human adult muscle nAChR influence channel function, I used two-electrode voltage clamp electrophysiology to perform a comprehensive mutant screen of all L9’ residues in each subunit of the human adult muscle nAChR. This resulted in a total of 76 unique mutations: 19 L9’ mutations consisting of every possible natural amino acid substitution in each subunit (α, β, ε, δ). The results of this screen show that while the polarity and size of a substituted residue contribute to its effect on channel function, increasing the polarity of the side chain typically has a more potentiating effect on channel function than does a change in size. The subunit in which the mutation is expressed also tailors the effect of a given mutation on channel function, with several δL9’ mutations producing qualitatively different effects than equivalent mutations in other subunits. Because the majority of L9’ mutations resulted in a gain-of-function, I originally postulated that interactions between L9’ and surrounding residues stabilize the resting state with the elimination of such interaction through mutations destabilizing the resting state to promote channel gating. Using a double mutant cycle, I explored interactions between the L9’ and adjacent non-L9’ residues but found that there are only weak or no interactions that contribute to channel function. Instead, my data support the hypothesis that the nAChR operates via a hydrophobic gating mechanism, and that adjacent L9’ residues are driven together by the hydrophobic effect to form a closed pore. L9’ mutations that either increase the polarity or decrease residue size likely reduce the hydrophobic driving forces that stabilizes the resting state, thus leading to an enhancement in channel function.

In an effort to search for a more selective, less toxic neuronal nicotinic acetylcholine receptor analgesic agent in comparison to epibatidine, a series of analogs with hybrid structures of epibatidine and ABT-594 were designed and synthesized. The 1-(pyridyloxymethyl)-7-azabicyclo[2.2.1]heptane ring systems were furnished via an intramolecular cyclization from a trans 1, 4 disubstitituted amino-cyclohexane derivative. The functionalized cyclohexane ring was formed via a [4+2] Diels-Alder cyclization reaction between the acetamidoacrylate and Danishefsky's diene. These 1- (pyridyloxymethyl)-7-azabicyclo[2.2.1]heptane ring systems were then tested in vitro as potential á4â2 nicotinic acetylcholine receptor ligands with high potency and selectivity. In addition, a series of rigid acetylcholine analogs were synthesized from cocaine to study the conformation of acetylcholine, the endogenous neurotransmitter at the nicotinic acetylcholine receptor. A stereoselective reduction of 2-tropinone led to the enantioselective synthesis of the desired acetoxytropane systems. These compounds were also tested in in vivo models for binding affinity and efficacy responses. Anabasamine, an alkaloid isolated from the Central Asian shrub, Anabasis aphylla, was synthesized for the first time. It was targeted due to interesting preliminary biological activity such as exhibiting anticholinesterase activity, anti-inflammatory activity, and facilitated an increase in hepatic alcohol dehydrogenase levels. Only preliminary studies were performed as anabasamine is limited in quantity due to its difficult isolation. A versatile synthetic methodology was developed for the synthesis of anabasamine and related nicotine analogs. This new methodology employed a pyridyl anion addition to valerolactone, for anabasamine, or butyrolactone for the nicotine analog, to afford 5-hydroxy-1-(6-methoxy-pyridin-3-yl)-pentan-1-one or 4-hydroxy-1-(6- methoxy-pyridin-3-yl)-butan-1-one, respectively. A reductive amination provided the piperidine ring moiety and a Suzuki coupling reaction introduced the bipyridyl moiety to anabasamine in five steps and 23% overall yield. In addition, this methodology was applied successfully to the synthesis of nicotine and other related analogs. In particular the synthesis of 6-methoxynicotine, a useful drug intermediate, was generated improving the yield from 16% over five steps to 54% over three steps.

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xii, 120 p.; also includes graphics. Includes bibliographical references (p. 110-120).

Thesis (Ph. D.)--University of Chicago. / Includes bibliographical references. Also available on the Internet.

Thesis (Honors)--College of William and Mary, 2010. / Includes bibliographical references (leaves 50-53). Also available online.

Pharmacotherapies for tobacco and alcohol cessation are only modestly successful, so it is important to better understand mechanisms underlying their use and abuse. The overarching goal of this research is to assess a6b2 subunit containing nicotinic acetylcholine receptor (a6b2*nAChR; *denotes possible assembly with other subunits) contributions to abuse-related effects of nicotine and alcohol. In the absence of a6b2*nAChR-selective agonists, a6b2*nAChR gain-of-function (a6L9’S) mice provide a tool for selective activation of a6b2*nAChRs. Using the a6L9’S mice together with nicotine doses sub-threshold for stimulation of native nAChRs, these studies tested the hypothesis that activation of a6b2*nAChRs is sufficient to promote neurochemical and behavioral effects relevant to nicotine addiction. Intracranial infusions of an a6b2*nAChR-selective antagonist further tested the neuroanatomical locus of a6b2*nAChR contributions to mesolimbic dopamine (DA) release and nicotine reward behavior. Our in vivo microdialysis and nicotine conditioned place preference (CPP) studies reveal that stimulation of a6b2*nAChRs on ventral tegmental area (VTA) DA neurons, as well as on DA terminals in the nucleus accumbens (NAc) shell support nicotine reward. VTA a6b2*nAChR stimulation is required for elevated basal NAc DA levels in a6L9’S mice, who also show elevated nicotine CPP. These studies also showed elevated anxiety-like behavior in a6L9’S mice, but no change in a6 subunit null mutant (a6KO) mice to suggest that elevated cholinergic tone at a6b2*nAChRs promotes anxiety-like behavior. To better define the molecular make-up of a6b2*nAChRs supporting nicotine reward and anxiety-like behavior, these studies crossed a6L9’S to a4 subunit knockout mice to differentiate (non-a4)a6b2* and a4a6b2*nAChR contributions. (non-a4)a6b2*nAChRs appear to promote nicotine reward behavior, while the a6b2*nAChR subtype that regulates anxiety-like behavior depends on the anxiety assay. Finally, these studies developed a mouse model of oral operant ethanol (EtOH) self-administration and assessed EtOH reinforcement in a6 heterozygous (a6HET) and a6KO mice to characterize the role of a6b2*nAChRs in EtOH reinforcement. EtOH self-administration was similar to wild type mice in a6KO mice, but not a6HET mice, suggesting that expression of a6b2*nAChRs modulates EtOH reinforcement. Together, these preclinical studies implicate a6b2*nAChRs in various abuse-related effects of nicotine and alcohol, identifying this receptor as a potential therapeutic target for treatment of dependence.

Prototoxin proteins have been identified as members of the Ly6/uPAR super family whose three-finger motif resembles that of α-bungarotoxin. Though they are known to modify the function of nAChRs, their specificity is still unclear. Our lab identified three prototoxin proteins in the chicken ciliary ganglion: Ch3ly, Ch5ly, and Ch6ly. Ch6ly was later identified as prostate stem cell antigen (PSCA), and specifically decreased the amount of calcium influx through the homomeric α7 nAChR subtype. I then identifiedCh3ly and Ch5ly as LY6E and LYPD6B, respectively. I focused my attention onLYPD6B because of its expression in the brain. This dissertation tests whether LYPD6Bis a prototoxin protein that specifically co-localizes with and modifies the function of the heteromeric α3β4* nAChRs (the other nAChR subtype expressed in the chicken ciliary ganglia). In the first part of my dissertation I performed intracellular two-electrode voltage clamp on Xenopus oocytes co-expressing human LYPD6B and different stoichiometries of the α3β4* nAChR, these included two (α3)2(β4)3 withβ4−α3−β4−β4−α3 and β4−α3−β4−α3−β4 stoichiometries, two (α3)3(β4)2 with stoichiometries β4−α3−α3−β4−α3 and β4−α3−β4−α3−α3, two (α3β4)2(α5D)β4−α3−α5D−β4−α3 and β4−α3−β4−α3−α5D, and (α3β4)2(α5N) with stoichiometries β4−α3−α5N−β4−α3 and β4−α3−β4−α3−α5N. Concatemeric constructs are designed to link nAChR subunits, thus when translated it is done so as a single polypeptide. LYPD6Bincreased the acetylcholine (ACh) potency and desensitization rate, but decreased the maximum current response (Imax) for the (α3)3(β4)2 nAChR subtype. Yet, LYPD6Bonly decreased the Imax for the (α3β4)2α5 D-variant and not the N-variant (associated with increase nicotine consumption). For the second part of my dissertation, I determined if the expression of LYPD6B correlated with nAChRs in an activity dependent manner. Though LYPD6B mRNA expression correlates with nAChR subunit mRNA expression levels, it seemed to be independent of nAChR activity. To determine if fluorescent colocalization occurs between LYPD6B and a specific nAChR subtype, I genetically engineered LYPD6B to express a human influenza hemagglutinin (HA) epitope tag and cloned into a chicken retrovirus. LYPD6B was shown to co-localize only with the α3β4*heteromeric and not the homomeric α7 nAChRs, in a nAChR activity dependent manner. This study adds to the complexity of a prototoxin’s function by suggesting that the specificity is dependent on nAChR type and stoichiometry. It is the first in identifying a prototoxin protein, LYPD6B, which specifically modulates the function of the(α3)3(β4)2 and (α3β4)2(α5 D-variant) heteromeric nAChR subtypes. For the (α3β4)2(α5D-variant) nAChR subtype LYPD6B decreased the Imax. Such observation may be telling of a novel mechanism involved with nicotine dependence. For the(α3)3(β4)2 nAChR subtype LYPD6B increases its ACh sensitivity, desensitization rate, while decreasing Imax. Additionally, the co-localization of LYPD6B and α3β4* nAChRsin the lack of nAChR activity highlights the relevance of the functional effects α3β4*nAChRs exhibit due to LYPD6B. Such relevance may be the utilization of limiting Ach amounts.