Nicotine addiction, opioid use disorder, and COVID-19 have made lasting impacts on every aspect of society. These are complicated conditions, and studies in these fields will likely continue for decades, if not centuries. Here, we make contributions to each of these issues using electrophysiology and microscopy. The first chapter goes into the motivation behind this thesis and the major experiments I used in my graduate career. In the second chapter, we introduce a new amino acid into the mouse muscle nicotinic acetylcholine receptor in an attempt to understand the dynamics of receptor activation. In the third chapter, we continue the Lester lab’s work on the neuroscientific effects of menthol and how it plays a role in nicotine addiction. We found the binding site for menthol on the α4β2 nicotinic acetylcholine receptor, which continues our hypothesis that the neuroscientific effects of menthol are detrimental to cigarette smokers. Fortunately, partly because of our studies, mentholated nicotine products are being phased out of the United States. The fourth and fifth chapters investigate μ-opioid receptor trafficking, both the trafficking from the endoplasmic reticulum and endocytosis from the plasma membrane. Both of these events play a role in inducing opioid use disorder and increasing the danger of using opioids. We hope that these studies will help other researchers understand opioid use disorder and fight the opioid epidemic. Finally, we studied the effects of SARS-COV-2 proteins on epithelial sodium channels. These channels are important for regulating lung fluid levels where their improper function may cause pulmonary edema. Pulmonary edema has been observed in COVID-19 patients. Altogether, we believe that we have made meaningful impacts on these important health concerns in this thesis. We look forward to how the scientific communities continue to build on our results.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Lester, Henry A.}, } @phdthesis{10.7907/Z9TT4P0Q, author = {Wong, Betty Ko}, title = {Biophysical Studies of Ligand-gated Ion Channels}, school = {California Institute of Technology}, year = {2017}, doi = {10.7907/Z9TT4P0Q}, url = {https://resolver.caltech.edu/CaltechTHESIS:05302017-225018783}, abstract = {This dissertation describes building a methodology for and the biophysical studies of ligand-gated ion channels (LGICs).
The primary focus of the first half of this dissertation is on developing a fluorescence-based assay to broadly study LGICs. Chapter 2 describes the site-selective incorporation of a turn-on fluorophore via unnatural amino acid mutagenesis on the mouse muscle-type nicotinic acetylcholine receptor (nAChR) in Xenopus oocytes as a proof-of-principle study. This method has proven to yield very low levels of undesired fluorescent background, which was a problem for previous incorporation techniques. Chapter 3 describes efforts towards imaging this in vivo system using lifetime imaging with efforts hampered by the inability to detect a clear signal. Chapter 4 describes efforts to apply the lifetime imaging approach towards a different system involving 5-HT3 proteins fused to fluorescent proteins in COS-7 cells.
The second half of this dissertation focuses on studies of menthol, a flavorant added to cigarettes that contributes to smoking addiction, as a negative allosteric modulator of the αβ42 nAChR. Chapter 5 reveals the stereochemical effects, or rather lack of, of menthol on the two stoichiometries of the αβ42 receptor. Chapter 6 seeks to identify the residue interactions with menthol of the αβ42 receptor using a combination of computational and experimental studies.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Dougherty, Dennis A.}, } @phdthesis{10.7907/Z9JD4TQ5, author = {Wall, Teagan Rose}, title = {Effects of TI-299423 on Neuronal Nicotinic Acetylcholine Receptors}, school = {California Institute of Technology}, year = {2015}, doi = {10.7907/Z9JD4TQ5}, url = {https://resolver.caltech.edu/CaltechTHESIS:03262015-100311493}, abstract = {Nicotinic acetylcholine receptors (nAChRs) are pentameric, ligand-gated, cation channels found throughout the central and peripheral nervous system, whose endogenous ligand is acetylcholine, but which can also be acted upon by nicotine. The subunit compositions of nAChR determine their physiological and pharmacological properties, with different subunits expressed in different combinations or areas throughout the brain. The behavioral and physiological effects of nicotine are elicited by its agonistic and desensitizing actions selectively on neuronal nAChRs. The midbrain is of particular interest due to its population of nAChRs expressed on dopaminergic neurons, which are important for reward and reinforcement, and possibly contribute to nicotine dependence. The α6-subunit is found on dopaminergic neurons but very few other regions of the brain, making it an interesting drug target. We assayed a novel nicotinic agonist, called TI-299423 or TC299, for its possible selectivity for α6-containing nAChRs. Our goal was to isolate the role of α6-containing nAChRs in nicotine reward and reinforcement, and provide insight into the search for more effective smoking cessation compounds. This was done using a variety of in vitro and behavioral assays, aimed dually at understanding TI-299423’s exact mechanism of action and its downstream effects. Additionally, we looked at the effects of another compound, menthol, on nicotine reward. Understanding how reward is generated in the cholinergic system and how that is modulated by other compounds contributes to a better understand of our complex neural circuitry and provides insight for the future development of therapeutics.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Lester, Henry A.}, } @phdthesis{10.7907/Z9HM56DT, author = {Parker, Rell Lin}, title = {Lynx1 Modulation of Nicotinic Acetylcholine Receptors}, school = {California Institute of Technology}, year = {2015}, doi = {10.7907/Z9HM56DT}, url = {https://resolver.caltech.edu/CaltechTHESIS:06292014-232713483}, abstract = {Nicotinic receptors are the target of nicotine in the brain. They are pentameric ion channels. The pentamer structure allows many combinations of receptors to be formed. These various subtypes exhibit specific properties determined by their subunit composition. Each brain region contains a fixed complement of nicotinic receptor subunits. The midbrain region is of particular interest because the dopaminergic neurons of the midbrain express several subtypes of nicotinic receptors, and these dopaminergic neurons are important for the rewarding effects of nicotine. The α6 nicotinic receptor subunit has garnered intense interest because it is present in dopaminergic neurons but very few other brain regions. With its specific and limited presence in the brain, targeting this subtype of nicotinic receptor may prove advantageous as a method for smoking cessation. However, we do not fully understand the trafficking and membrane localization of this receptor or its effects on dopamine release in the striatum. We hypothesized that lynx1, a known modulator of other nicotinic receptor subtypes, is important for the proper function of α6 nicotinic receptors. lynx1 has been found to act upon several classes of nicotinic receptors, such as α4β2 and α7, the two most common subtypes in the brain. To determine whether lynx1 affects α6 containing nicotinic receptors we used biochemistry, patch clamp electrophysiology, fast scan cyclic voltammetry, and mouse behavior. We found that lynx1 has effects on α6 containing nicotinic receptors, but the effects were subtle. This thesis will detail the observed effects of lynx1 on α6 nicotinic receptors.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Lester, Henry A.}, } @phdthesis{10.7907/Z96D5QZB, author = {Dilworth, Crystal Noelle}, title = {Fluorescence Microscopy of Nicotinic Acetylcholine Receptors}, school = {California Institute of Technology}, year = {2014}, doi = {10.7907/Z96D5QZB}, url = {https://resolver.caltech.edu/CaltechTHESIS:05282014-200718606}, abstract = {
Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels abundantly expressed in the central nervous system. Changes in the assembly and trafficking of nAChRs are pertinent to disease states including nicotine dependence, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), and Parkinson’s disease (PD). Here we investigate the application of high resolution fluorescence techniques for the study of nAChR assembly and trafficking. We also describe the construction and validation of a fluorescent α5 subunit and subsequent experiments to elucidate the cellular mechanisms through which α5 subunits are expressed, assembled into mature receptors, and trafficked to the cell surface. The effects of a known single nucleotide polymorphism (D398N) in the intracellular loop of α5 are also examined.
Additionally, this report describes the development of a combined total internal reflection fluorescence (TIRF) and lifetime imaging (FLIM) technique and the first application of this methodology for elucidation of stochiometric composition of nAChRs. Many distinct subunit combinations can form functional receptors. Receptor composition and stoichiometry confers unique biophysical and pharmacological properties to each receptor sub-type. Understanding the nature of assembly and expression of each receptor subtype yields important information about the molecular processes that may underlie the mechanisms through which nAChR contribute to disease and addiction states.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Lester, Henry A.}, } @phdthesis{10.7907/2S9B-R910, author = {Nichols, Weston A.}, title = {Lynxl and the β2V287L Mutation Affect the Stoichiometry of the α4β2 Nicotinic Acetylcholine Receptor}, school = {California Institute of Technology}, year = {2014}, doi = {10.7907/2S9B-R910}, url = {https://resolver.caltech.edu/CaltechTHESIS:05292014-190146869}, abstract = {GPI-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on α4β2 nAChRs within the endoplasmic reticulum. Lynx affects assembly of nascent α4 and β2 subunits, and alters the stoichiometry of the population that reaches the plasma membrane. Additionally, these data suggest that lynx1 alters nAChR stoichiometry primarily through this intracellular interaction, rather than via effects on plasma membrane nAChRs. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any GPI-anchored protein, could act within the cell to alter assembly of multi-subunit protein.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Lester, Henry A.}, } @phdthesis{10.7907/4AGK-FP05, author = {Frazier, Shawnalea Jimee}, title = {Optimization of the GluC1/IVM Neuronal Silencing Tool via Protein Engineering}, school = {California Institute of Technology}, year = {2012}, doi = {10.7907/4AGK-FP05}, url = {https://resolver.caltech.edu/CaltechTHESIS:05252012-121406958}, abstract = {A variety of genetically encoded tools have been developed for deciphering the neural circuitry of the brain. Such tools allow physical manipulation of neuronal excitability in a reversible, cell-specific manner, enabling researchers to establish how electrical activity and connectivity facilitate the information processing that mediates perception and drives behavior. An expanding toolkit of engineered neuroreceptors, particularly those actuated by orthogonal pharmacological ligands, provide noninvasive manipulation of regional or disperse neuronal populations with adequate spatiotemporal precision and great potential for multiplexing. We previously engineered an invertebrate glutamate-gated chloride channel (GluCl αβ) that enabled pharmacologically induced silencing of electrical activity in targeted CNS neurons in vivo by the anthelmintic drug compound ivermectin (IVM; Lerchner et al., 2007). With this receptor, GluCl opt α-CFP + opt β-YFP Y182F, the concentration of IVM necessary to elicit a consistent silencing phenotype was higher than expected, raising concern about its potential side effects. Considerable variability in the extent of spike suppression was also apparent and was attributed to variable co-expression levels of α and β subunits. Thus, a rational protein engineering strategy was employed to optimize the GluCl/IVM tool. To increase agonist sensitivity, a gain-of-function gating mutation involving the highly conserved leucine 9’ residue of the α pore-lining M2 transmembrane domain was introduced. Various mutations at this position facilitate channel opening in the absence and presence of ligand. Analysis of side chain properties revealed that helix-destabilizing energy correlated with increases in agonist sensitivity. One mutation, L9’F, enhances β subunit incorporation to substantially increase IVM sensitivity without permitting unliganded channel opening. Removal of an arginine-based ER retention motif (RSR_AAA) from the intracellular loop of β promoted plasma membrane expression of heteromeric GluCl αβ by preventing ER-associated degradation of the β subunit. An additional monomeric XFP mutation complements these effects. The newly engineered GluCl opt α-mXFP L9’F + opt β-mXFP Y182F RSR_AAA receptor significantly increases conductance and reduces variability in evoked spike generation in vitro using a lower concentration of IVM. This receptor, dubbed ‘GluClv2.0’, is an improved tool for IVM-induced silencing.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Lester, Henry A.}, } @phdthesis{10.7907/AGAJ-PE93, author = {Wade, Lawrence A.}, title = {An Evanescent Perspective on Cells}, school = {California Institute of Technology}, year = {2011}, doi = {10.7907/AGAJ-PE93}, url = {https://resolver.caltech.edu/CaltechTHESIS:09102010-145946974}, abstract = {We have optically sectioned living cells to a maximum depth of ~250 nm using a Variable Angle-Total Internal Reflection Fluorescence Microscope (VA-TIRFM). This yields 3D images of cell membranes and nearby organelles similar to that gained by confocal microscopes but with at least an order-of-magnitude greater depth resolution. It also enables cellular membranes to be imaged in near isolation from cell organelles. Key to achieving this resolution was integration of a controllable excitation laser micropositioner into a standard through-the-lens TIRF illuminator and development of a custom culture dish for re-use of expensive high index of refraction cover slips. Images are acquired at several penetration depths by varying the excitation laser illumination angles. At the shallowest penetration depth (~46 nm) just the membrane and a few internal puncta are imaged. As the penetration depth is increased up to 250 nm organelles near the membrane, such as the ER, are imaged as well. The sequence of images from shallow deep is processed to yield a z-stack of images of approximately constant thickness at increasing distance from the coverslip. We employ this method to distinguish membrane-localized fluorophores (α4 GFP β2 nicotinic acetylcholine receptors and pCS2:lyn-mCherry) at the plasma membrane (PM) from those in near-PM endoplasmic reticulum (ERTracker green, α4 GFP β2 nicotinic acetylcholine receptors), on a z-axis distance scale of ~45 to ~250 nm in N2a cells. In doing so we observe occasional smooth ER structures that cannot be resolved as being distinct from the membrane.
In a second project substantial progress has been made towards developing a Tip Enhanced Fluorescence Microscope (TEFM) capable of imaging wet biological samples with ~10 nm resolution. A TEFM combines a TIRFM with an Atomic Force Microscope (AFM) to modulate sample fluorescence through near-field dipole-dipole coupling.
In the third project the capability to consistently produce high quality nanotube AFM probes was developed and a technique for chemically functionalizing the tip of a nanotube AFM probe was invented.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Fraser, Scott E.}, } @phdthesis{10.7907/17AK-6J11, author = {Bower, Kiowa San}, title = {Chemical-Scale Studies of the 5-HT₃ and D2 Dopamine Receptors}, school = {California Institute of Technology}, year = {2010}, doi = {10.7907/17AK-6J11}, url = {https://resolver.caltech.edu/CaltechTHESIS:03142010-163350106}, abstract = {
During synaptic transmission in the central nervous system, neuroreceptors transduce a chemical signal into an electrical signal, a process that is mediated by both ligand-gated ion channels (LGICs) and G-protein coupled receptors (GPCRs). The work in this thesis examines structure-function relationships within these receptors, with a focus on elucidating the mechanism of molecular recognition during ligand binding. We utilize conventional and unnatural amino acid mutagenesis, structural derivatives of agonists, and homology models to identify specific interactions and the role of binding site residues in ligand binding and receptor activation. The technique of unnatural amino acid mutagenesis allows us to study these processes in greater detail than would otherwise be possible, even at the scale of a chemical bond.
Chapter 2 covers structure-function investigations of a ligand-gated ion channel, the 5-HT₃ receptor, with a goal of understanding agonist binding and receptor activation. The project examines residues in close proximity to the ligand-binding site and focuses on polar interactions with hydrophilic residues. We identify 5-fluorotryptamine (5-FT) as a partial agonist of the 5-HT₃ receptors and show that size and electronegativity are important at the 5’ position for efficient channel opening. Our investigation of the compound 1-OT revealed it to be an agonist of equal potency to the native agonist (5-HT), demonstrating that the indolic proton of serotonin is not essential to its activation of the receptor. A study focusing on loop A residues led us to refine our homology model and propose that Glu129 faces into the binding pocket, where, through its ability to hydrogen bond, it plays a critical role in ligand binding. Further studies of binding site residues identified an ionic interaction that likely participates in the conformational changes associated with receptor gating and characterized several other residues that play critical roles in receptor activation. Finally, we compare and contrast the behaviors of two structurally distinct agonist classes, 5-HT and its related structures, and m-chlorophenylbiguanide (mCPBG) and identify several residues that play critical roles in modulating agonist binding and gating in response to these agonists.
Chapter 3 describes a study examining the binding site and the mechanism of agonist activation of a GPCR, the D2 dopamine receptor. A number of aromatic amino acids thought to be near the agonist binding site were evaluated. Incorporation of a series of fluorinated tryptophan derivatives at a conserved tryptophan of the D2 receptor establishes a cation-π interaction between the agonist dopamine and this residue (W6.48), suggesting a reorientation of W6.48 on agonist binding, consistent with proposed “rotamer switch” models.
Finally, chapter 4 describes a project that seeks to extend the nonsense suppression methodology to include mammalian expression systems. Progress is made developing techniques for efficient transfection of cells in culture.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Dougherty, Dennis A.}, } @phdthesis{10.7907/3Q8S-CV89, author = {Imoukhuede, Princess Ikhianosen Uerenikhosen}, title = {Visualizing the Membrane Confinement, Trafficking and Structure of the GABA Transporter, GAT1}, school = {California Institute of Technology}, year = {2008}, doi = {10.7907/3Q8S-CV89}, url = {https://resolver.caltech.edu/CaltechETD:etd-03142008-155402}, abstract = {
Transporter trafficking regulators can play an important role in maintaining the transporter density necessary for effective function. I determine interactions that confine GAT1 at the membrane by investigating GAT1 lateral mobility through fluorescence recovery after photobleaching (FRAP). I find that the mobility of GAT1 can be increased by depolymerizing actin or by blocking the GAT1 PDZ interacting domain. I also identify ezrin as the GAT1 adaptor to actin. Through fluorescence resonance energy transfer (FRET), the distance between GAT1-YFP and Ezrin-CFP is calculated as 64–68 Å, and it can be significantly increased by disrupting the actin cytoskeleton. Altogether, my data reveals that actin confines GAT1 to the plasma membrane via ezrin, an interaction mediated through the GAT1-PDZ interaction domain.
Discoveries in the field of vesicle fusion provide direct ties to translational research. While the study of vesicle fusion classically has been applied to neurotransmitter and neuropeptide containing vesicles; there is evidence that secretory vesicles physiologically differ from vesicles trafficking membrane protein. For instance, GAT1 resides on a vesicle lacking neurotransmitter but containing some v-SNARE proteins. These differences in the vesicle composition suggest inherent differences in trafficking mechanisms, which can only be confirmed through further study of membrane protein trafficking. To this end, I apply total internal reflection fluorescence microscopy (TIRFM) to quantify the number of GAT1 molecules on vesicles and to observe the movement of vesicles containing fluorescently tagged GAT1 into the plasma membrane. I determine that these vesicles contain 3–7 molecules of GAT1 and uncover a population of GAT1 vesicles with ATP-dependent lateral displacement.
The protein-protein interactions, trafficking, and oligomerization of mouse GAT1 were studied using fourteen different fusions of mGAT1 with fluorescent protein. We determine that a natural PDZ-interacting motif is minimally required for wild-type GAT1 behavior. Fusions with wild-type function yielded up to 21% FRET efficiency, indicating efficient GAT1 oligomerization. Additionally, 45% FRET was observed between a GAT1 construct and YFP-syntaxin-1A. Inserting XFP between R565 and L566, resulted in 33% FRET but impaired function, which indicated the “RL” motif in the proximal C terminus governs export from the endoplasmic reticulum but not transporter oligomerization.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Lester, Henry A.}, } @phdthesis{10.7907/RYJJ-JW20, author = {Slimko, Eric Michael}, title = {Selective Silencing of Vertebrate Neurons: Strategies Using Invertebrate Ligand-Gated Ion Channels}, school = {California Institute of Technology}, year = {2006}, doi = {10.7907/RYJJ-JW20}, url = {https://resolver.caltech.edu/CaltechETD:etd-05142008-004518}, abstract = {Selectively reducing the excitability of specific neurons will (1) allow for the creation of animal models of certain human neurological disorders and (2) provide insight into the roles of specific sets of neurons, both in the local circuit and in the behavior of the intact organism. This work focuses on a combined genetic and pharmacological approach to silence neurons electrically. We express invertebrate ivermectin (IVM)-sensitive chloride channels (Caenorhabditis elegans GluCl α and β) in vertebrate neurons first in vitro using viral and tranfection techniques, and then finally in vivo using genetic techniques, to produce inhibition via a Cl- conductance when activated with IVM. We have considerably engineered these two genes by (1) re-coding the genes such that vertebrate-preferred codons are used throughout the sequences, (2) incorporating fluorescent tags within the proteins, and (3) finding a mutation to remove the undesirable glutamate sensitivity of the channel while retaining IVM efficacy. Expression of this new channel does not affect the normal spike activity of the target cell, yet the experimentor can effectively “shut-off” the cell with concentrations of as low as 5 nM IVM. Chapter 1 provides a broad overview of the many “selective silencing” approaches that experimenters have tried. In Chapter 2, the author describes the basic “GluCl/IVM” technique and initial experiments in cultured hippocampal neurons. Chapter 3 refines the technique by describing the strategy and mutation that allowed great reduction in the native glutamate response while maintaining the IVM response. Chapter 4 develops the final engineering of the channel: recoding the sequence for optimal expression and the introduction of fluorescent tags for identification. Finally, Chapters 5 and 6 discuss the successes and failures of in vivo work with what we now call the “GluCl/IVM method.”}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Lester, Henry A.}, } @phdthesis{10.7907/XZ69-9A14, author = {Shapovalov, George G.}, title = {Mechanosensitive Channels of Bacteria: Structure and Function. Electrophysiology as a High Resolution Technique of Ion Channel Study}, school = {California Institute of Technology}, year = {2005}, doi = {10.7907/XZ69-9A14}, url = {https://resolver.caltech.edu/CaltechETD:etd-12122004-215151}, abstract = {Mechanosensitive (MS) ion channels commonly play a role of transducers converting mechanical stimuli into electrical or chemical signaling, thus allowing the cell to regulate its behavior in response to changing environment conditions. MS channels participate in sensation of sound and orientation in inner ear (hair cells), in touch sensation and in osmoregulation of bacteria. Structure of bacterial MS channels of large (MscL) and small (MscS) conductance has been recently solved at atomic resolution, stimulating various structural and functional studies. In this work author presents series of experiments enhancing an understanding of mechanosensation in bacteria. In Chapter 2 author performs cysteine cross-linking experiments suggesting asymmetric gating pattern of Tb-MscL ion channel. Chapters 3 and 4 establish a possibility of successful synthesis of fully functional Tb- and Ec-MscL proteins displaying a phenotype identical to recombinant channels. Studies in Chapter 5 and Appendix 1 extend the resolution of single-channel patch clamping technique, and describe a fine structure of MS channel gating by collecting and characterizing intersubstate transitions.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Lester, Henry A.}, } @phdthesis{10.7907/y4ay-ph28, author = {Zacharias, Niki Marie}, title = {Chemical-Scale Manipulation of Ion Channels: in vivo Nonsense Suppression and Targeted Disulfide Crosslinking}, school = {California Institute of Technology}, year = {2004}, doi = {10.7907/y4ay-ph28}, url = {https://resolver.caltech.edu/CaltechETD:etd-01042004-210542}, abstract = {The study of the three-dimensional shape and structure-function relationships of ion channels is a very challenging field of research. Ion channels are integral-membrane proteins that when open allow ions to flux across the cell membrane. The structure and function of ion channels are dependent on the cell membrane that surrounds them. Because an ion channel must be embedded in a cell membrane, many techniques used to probe the structure of soluble proteins cannot be used in the study of ion channels.
One versatile technique that has been shown to be quite valuable in the structure-function studies of ion channels is the in vivo nonsense suppression method for unnatural amino acid incorporation. This technique allows one to site-specifically incorporate an unnatural amino acid or hydroxy acid into a protein in a living cell. To date more than 60 amino acids and hydroxy acids have been incorporated into proteins using in vivo nonsense suppression. The method has been shown to accommodate a wide variety of unnatural amino acids and hydroxy acids. Chapter One will discusses the in vivo nonsense suppression method in greater detail.
A key component of this work is the design and synthesis of new unnatural amino acids that have novel properties. Chapter 2 discusses the synthesis and uses of 5-(o-nitrobenzyl)selenyl-2-hydroxypentanoic acid (NBSeOH). NBSeOH is used to site-specifically cleave a peptide backbone. The o-nitrobenzyl protecting group is photochemically removed to reveal a selenium anion. The selenium anion then initiates an intramolecular SN2 displacement that cleaves the backbone of the protein. Preliminary data reveals that NBSeOH can be incorporated into a protein in vivo and in vitro, and photolysis of proteins and peptides containing NBSeOH does lead to protein backbone cleavage.
Chapter 4 discusses how the in vivo nonsense suppression method was used to incorporate unnatural amino acids containing a quaternary ammonium moiety to mimic the quaternary ammonium on acetylcholine. These unnatural amino acids were used to probe the nicotinic acetylcholine receptor?s binding site. These unnatural amino acids are called tethered agonists because when they were incorporated into four different positions on the nicotinic acetylcholine receptor partial opening of the channel occurred even when agonist was not present. These tethered agonists were used to obtain distance information about where acetylcholine binds within the receptor.
Another technique used to probe the structure of ion channels is targeted disulfide crosslinking. In the targeted disulfide crosslinking method, cysteine residues are introduced at various locations throughout a protein and oxidized to see whether disulfide bond formation can occur. Since only cysteine residues close in space will form a disulfide bond, this method can reveal fine structural aspects of a protein. The method was used to study the pore lining structure of the nicotinic acetylcholine receptor. Several cysteine mutants were made using mutagenesis and then studied in functional channels expressed in Xenopus oocytes. The channels were then exposed to oxidizing agents, and the ability of these mutant channels to form disulfide bonds was evaluated. Chapter 3 describes the work dealing with the targeted disulfide crosslinking experiments in the nicotinic acetylcholine receptor.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Dougherty, Dennis A.}, } @phdthesis{10.7907/e390-k051, author = {Figl, Antonio}, title = {Structure-Function Analysis of the β Subunit of Neuronal Nicotinic Acetylcholine Receptors}, school = {California Institute of Technology}, year = {1996}, doi = {10.7907/e390-k051}, url = {https://resolver.caltech.edu/CaltechTHESIS:11072019-170728389}, abstract = {Nicotinic receptors belong to the superfamily of ligand-gated ion channels. Since evidence was rapidly accumulating implicating the non-α subunits in ligand-binding events, we decided to investigate eventual contributions of the neuronal β subunit to these events by performing a series of increasingly detailed experiments on a series of chimeric β subunits. In the first set of experiments, we constructed a variety of chimeric β subunits consisting of NH2-terminal neuronal β4 sequences and COOH-terminal β2 sequences and expressed them with the α3 subunit in Xenopus oocytes. The results showed that (a) two residues in the extracellular domain of chimeric β4•β2 subunits (108β2Phe↔︎β4Val, 110β2Ser↔︎β4Thr) account for much of the relative cytisine sensitivity; and (b) four extracellular residues of chimeric β4•β2 subunits (112β2Ala↔︎β4Val, 113β2Val↔︎β4Ile and 115β2Ser↔︎β4Arg, 116β2Tyr↔︎β4Ser) account for most of the relative tetramethylammonium sensitivity.
Encouraged by the above results, we continued our experiments with additional chimeras of the β2 and β4 neuronal nicotinic subunits to locate regions that contribute to differences between the acetylcholine dose-response relationships of α3β2 and α3β4 receptors. Substitutions within the first 120 residues convert the EC50 for ACh from one wild-type value to the other, suggesting that amino acids within the first 120 residues of β2 and the corresponding region of β4 contribute to an agonist binding site that bridges the α and β subunits in neuronal nicotinic receptors.
Since the EC50 phenotypes caused by the β2 and β4 subunits could be due to a difference in gating or binding properties, we attempted to unravel this question by performing voltage-jump relaxations for the series of neuronal nicotinic acetylcholine receptors we constructed previously. The chimeric β4/β2 subunits showed a transition in the concentration dependence of the relaxation rate constants in the region between residues 94 and 109, analogous to our previous observation with steady-state dose-response relationships. The data reinforce previous conclusions that the region between residues 94 and 109 on the β subunit plays a role in binding agonist but also show that other regions of the receptor control gating kinetics subsequent to the binding step.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Lester, Henry A.}, } @phdthesis{10.7907/eyre-8424, author = {McDonough, Stefan I.}, title = {Pharmacology and pore-forming domains of the cystic fibrosis transmembrane conductance regulator}, school = {California Institute of Technology}, year = {1994}, doi = {10.7907/eyre-8424}, url = {https://resolver.caltech.edu/CaltechTHESIS:05142013-132218781}, abstract = {The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel member of the ATP-binding cassette (ABC) superfamily of membrane proteins. CFTR has two homologous halves, each consisting of six transmembrane spanning domains (TM) followed by a nucleotide binding fold, connected by a regulatory (R) domain. This thesis addresses the question of which domains are responsible for Cl^- selectivity, i.e., which domains line the channel pore.
To address this question, novel blockers of CFTR were characterized. CFTR was heterologously expressed in Xenopus oocytes to study the mechanism of block by two closely related arylaminobenzoates, diphenylamine-2-carboxylic acid (DPC) and flufenamic acid (FFA). Block by both is voltage-dependent, with a binding site ≈ 40% through the electric field of the membrane. DPC and FFA can both reach their binding site from either side of the membrane to produce a flickering block of CFTR single channels. In addition, DPC block is influenced by Cl^- concentration, and DPC blocks with a bimolecular forward binding rate and a unimolecular dissociation rate. Therefore, DPC and FFA are open-channel blockers of CFTR, and a residue of CFTR whose mutation affects their binding must line the pore.
Screening of site-directed mutants for altered DPC binding affinity reveals that TM-6 and TM-12 line the pore. Mutation of residue 5341 in TM-6 abolishes most DPC block, greatly reduces single-channel conductance, and alters the direction of current rectification. Additional residues are found in TM-6 (K335) and TM-12 (T1134) whose mutations weaken or strengthen DPC block; other mutations move the DPC binding site from TM-6 to TM-12. The strengthened block and lower conductance due to mutation T1134F is quantitated at the single-channel level. The geometry of DPC and of the residues mutated suggest α-helical structures for TM-6 and TM-12. Evidence is presented that the effects of the mutations are due to direct side-chain interaction, and not to allosteric effects propagated through the protein. Mutations are also made in TM-11, including mutation S1118F, which gives voltage-dependent current relaxations. The results may guide future studies on permeation through ABC transporters and through other Cl^- channels.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Lester, Henry A.}, } @phdthesis{10.7907/svss-ye57, author = {Hsu, Hsiaolan S.}, title = {Properties of the first genetically engineered neuron.}, school = {California Institute of Technology}, year = {1993}, doi = {10.7907/svss-ye57}, url = {https://resolver.caltech.edu/CaltechTHESIS:12062012-091333466}, abstract = {
Electrically excitable channels were expressed in Chinese hamster ovary cells using a vaccinia virus vector system. In cells expressing rat brain IIA Na^+ channels, brief pulses (< 1ms) of depolarizing current resulted in action potentials with a prolonged (0.5-3s) depolarizing plateau; this plateau was caused by slow and incomplete Na^+ channel inactivation. In cells expressing both Na^+ and Drosophila Shaker H4 transient K^+ channels, there were neuron-like action potentials. In cells with appropriate Na+/K+ current ratios, maintained stimulation produced repetitive firing over a 10-fold range of frequencies but eventually led to “lockup” of the potential at a positive value after several seconds of stimulation; the latter effect was due primarily to slow inactivation of the K^+ currents. Numerical simulations of modified Hodgkin-Huxley equations describing these currents, using parameters from voltage-clamp kinetics studied in the same cells, accounted for most features of the voltage trajectories. The present study shows that insights into the mechanisms for generating action potentials and trains of action potentials in real excitable cells can be obtained from the analysis of synthetic excitable cells that express a controlled repertoire of ion channels. This model system provides a direct control of complexity of neuronal behavior, and a tool for studying various forms of neural modulation at molecular and cellular levels.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Lester, Henry A.}, } @phdthesis{10.7907/fj5c-4h83, author = {Yu, Lei}, title = {The Nicotinic Acetylcholine Receptor: Gene Expression and Ion Channel Function}, school = {California Institute of Technology}, year = {1987}, doi = {10.7907/fj5c-4h83}, url = {https://resolver.caltech.edu/CaltechTHESIS:10312019-114338923}, abstract = {The nicotinic acetylcholine receptor (AChR) is a complex protein, which functions as a ligand-gated ion channel on the postsynaptic membrane at the neuromuscular junction and mediates signal transmission from neuron to muscle. Research on the AChR has had a long history and has benefited from the endeavors of scientists from many disciplines. The intensive, multidisciplinary studies have yielded valuable knowledge about this molecule, which serves as a model for the understanding of many fundamental questions in biological sciences. Chapter 1 presents a review of the AChR.
As a tissue-specific and developmental stage-specific molecule, AChR is under temporal and spatial control for its synthesis. Chapter 2 reports a qualitative and quantitative study of AChR gene activity during muscle cell differentiation, using a cDNA clone isolated from a murine muscle cell line, which codes for the γ subunit of the mouse AChR. The results indicate that the regulation of mRNA accumulation levels is a major mechanism in the differential synthesis of the AChR.
The marriage between AChR and molecular biology resulted in many cDNA clones which, after being introduced to African frogs, produced the next generation — Xenopus oocytes with exotic AChRs on them. Chapter 3 describes the attempt to localize “determinants” that specify species subunit identity in the AChR by constructing chimeric cDNA clones composed of fragments from different origins, taking advantage of the Xenopus oocyte expression system. The results from surface toxin-binding assay and two-electrode voltage-clamp recording suggested that while the species specificity can be dictated by certain subunits, the determination of subunit identity does not reside at a defined locus in the fragments tested.
Does the complex composition of multisubunits in the AChR bear any functional significance? Chapter 4 addresses this question through the study of mouse-Torpedo AChR hybrids. The complete substitution of AChR subunits between mouse and Torpedo receptors generated all 16 combinations, and a systematic analysis of these hybrids revealed an interesting pattern with respect to the voltage sensitivity in the ACh-induced response: The identity of the β subunit determines, while the interaction between the β and δ subunits modulates, the AChR voltage sensitivity. The results, therefore, suggest that different subunits of AChR may play a central role in different functional properties.
Patch-clamp technique has offered an opportunity for analyzing transmembrane current flow with the high resolution of single-channel recording. Chapter 5 describes such a study on homologous and hybrid AChRs. Voltage influence on the three parameters were evaluated, and the results indicate that the single channel conductance is independent of membrane potential and that the channel closing and opening rates together constitute the basis for the voltage sensitivity in whole-cell recording with the closing rate making the major contribution. Also investigated were the subunit roles in species specificity of channel-open duration and voltage dependence. The results are in agreement with those reported on channel duration and support the conclusions of our previous work on the subunit involvement in determining the voltage sensitivity of the AChR response.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Davidson, Norman R.}, } @phdthesis{10.7907/wb48-ph26, author = {Krouse, Mauri Eugene}, title = {Investigation of Competitive Antagonist Binding to the Nicotinic Acetylcholine Receptor Using Voltage-Jump and Light-Flash Techniques}, school = {California Institute of Technology}, year = {1984}, doi = {10.7907/wb48-ph26}, url = {https://resolver.caltech.edu/CaltechTHESIS:05282015-161004818}, abstract = {
NOTE: Text or symbols not renderable in plain ASCII are indicated by […]. Abstract is included in .pdf document.
The kinetics of curare inhibition were measured at the postsynaptic membrane of frog sartorius and cutaneous pectoris muscle fibers. Acetyleholine (ACh) and d-tubocurarine (dTC) were iontophoresed from twin-barrel micropipettes, and the muscle fiber’s membrane potential was recorded intracellularly. By itself dTC produced no change in membrane potential, but dTC-receptor binding was assayed by observing changes in the response to a constant pulse of ACh.
The responses to both ACh and dTC had brief latencies, reached their maxima rapidly, and were highly sensitive to the dose. Under these conditions, the kinetics of drug action are not slowed by access of the drugs to the synaptic cleft.
After a pulse of dTC, recovery from inhibition proceeds slowly along an exponential time course with a rate constant, […]. The recovery rate does not depend on the maximal level of inhibition and varies only slightly with temperature (Q10 = 1.25).
After a sudden maintained increase in dTC release, inhibition develops approximately exponentially until a steady-state level of inhibition is reached. The apparent rate constant for the onset of inhibition, […], is greater than […]. When the steady-state inhibition reduces the ACh response to 1/n of its control value, […] = […].
When the ACh sensitivity is reduced with cobra toxin, both […] and […] increase. Thus, the kinetics of dTC inhibition depend on the density of acetyleholine receptors in the synaptic cleft. If the density of acetylcholinesterase is reduced in the cleft by collagenase, […] increases only twofold.
When the nerve terminal is removed after collagenase action, and the drugs are iontophoresed directly onto the exposed postsynaptic membrane, […] increases more than tenfold.
Bath-applied dTC competitively inhibits the responses to brief iontophoretic ACh pulses with an apparent equilibrium dissociation constant, KD = 0.5 […]. This suggests that dTC molecules equilibrate with the receptors on a millisecond time scale.
On denervated frog muscle cells, extrasynaptic receptors have a lower apparent affinity for dTC. After a pulse of dTC, inhibition decays tenfold more rapidly at these extrasynaptic sites than at the nerve-muscle synapse.
It is suggested that dTC inhibits synaptic receptors more effectively because the nerve terminal restricts diffusion within the synaptic cleft and each dTC molecule binds repeatedly to several acetylcholine receptors before escaping from the cleft. Consequently, the receptors transiently buffer the concentration of dTC in the cleft, and the macroscopic kinetics of inhibition are much slower than the molecular rates of dTC binding.