Charles Luetje - US grants

[unreadable] DESCRIPTION (provided by applicant): The goals of this project are: (1) to elucidate the structure of the site(s) on neuronal nicotinic acetylcholine receptors (nAChRs) where zinc binds and potentiates receptor function, and (2) to elucidate the mechanism through which zinc potentiates receptor function. [unreadable] Nicotinic ligands are potentially useful as anxiolytics and analgesics, in the treatment of neurological disorders such as schizophrenia, Parkinson's disease, and Alzheimer's disease, and are also the sites at which nicotine exerts its psychoactive and addictive effects. Effective pharmacological intervention at neuronal nAChRs requires development of subtype selective ligands. Pursuit of this goal has traditionally been directed toward development of ligands that act at the acetylcholine (ACh) binding sites. We propose to pursue a new class of site on neuronal nAChRs, the site at which zinc potentiates these receptors. The ACh binding sites on neuronal nAChRs are at the interfaces between the extracellular domains of subunits. In many neuronal nAChRs, two agonist-binding sites are formed at interfaces between two pairs of dissimilar subunits, leaving three alternate interfaces with no involvement in ACh binding. We hypothesize that one or more of the alternate interfaces bind zinc, a modulator of neuronal nAChR function. In aim 1, we will use site-directed mutagenesis and functional analysis to identify amino acid residues on neuronal nAChRs that coordinate zinc at the potentiation site. In aim 2, we will explore the larger structure of the potentiation site using Substituted Cysteine Accessibility Mutagenesis. Information obtained in aims 1 and 2 will be used to refine a homology model of the extracellular domains of neuronal nAChRs. In aim 3, we will determine whether zinc potentiates neuronal nAChRs through changes in the single channel open probability, the single channel current, and/or the number of channels. Changes in open probability will be characterized in terms of the underlying gating mechanism. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The goals of this project are: (1) to elucidate the structure of the site(s) on neuronal nicotinic acetylcholine receptors (nAChRs) where zinc binds and potentiates receptor function, and (2) to elucidate the mechanism through which zinc potentiates receptor function. [unreadable] Nicotinic ligands are potentially useful as anxiolytics and analgesics, in the treatment of neurological disorders such as schizophrenia, Parkinson's disease, and Alzheimer's disease, and are also the sites at which nicotine exerts its psychoactive and addictive effects. Effective pharmacological intervention at neuronal nAChRs requires development of subtype selective ligands. Pursuit of this goal has traditionally been directed toward development of ligands that act at the acetylcholine (ACh) binding sites. We propose to pursue a new class of site on neuronal nAChRs, the site at which zinc potentiates these receptors. The ACh binding sites on neuronal nAChRs are at the interfaces between the extracellular domains of subunits. In many neuronal nAChRs, two agonist-binding sites are formed at interfaces between two pairs of dissimilar subunits, leaving three alternate interfaces with no involvement in ACh binding. We hypothesize that one or more of the alternate interfaces bind zinc, a modulator of neuronal nAChR function. In aim 1, we will use site-directed mutagenesis and functional analysis to identify amino acid residues on neuronal nAChRs that coordinate zinc at the potentiation site. In aim 2, we will explore the larger structure of the potentiation site using Substituted Cysteine Accessibility Mutagenesis. Information obtained in aims 1 and 2 will be used to refine a homology model of the extracellular domains of neuronal nAChRs. In aim 3, we will determine whether zinc potentiates neuronal nAChRs through changes in the single channel open probability, the single channel current, and/or the number of channels. Changes in open probability will be characterized in terms of the underlying gating mechanism. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Mammals can distinguish among thousands of odorants and the first step in this process is the interaction between odorant ligand and odorant receptor (OR). The mammalian odorant receptors constitute an enormous family of G-protein coupled receptors (GPCRs). Sequence analysis has grouped these receptors into two broad classes, with each class further divided into many subfamilies. Several studies, including our preliminary work, indicate that while the members of an OR subfamily recognize similar chemical structures, they can distinguish among these ligands. This project is directed toward understanding how ORs recognize and distinguish among ligands at the molecular level. We have developed a functional assay for ORs using the Xenopus oocyte expression system and robotic electrophysiology. In Aim 1, we will screen representative ORs from a broad range of subfamilies to identify new ligand - OR pairings. In Aim 2, we will then clone and functionally express other members of the particular OR subfamilies. Detailed pharmacological analysis will be conducted to reveal differences in ligand specificity among subfamily members. In our third and fourth specific aims, we will combine computational homology modeling and ligand docking with site-directed mutagenesis and functional analysis to identify the structural basis for differences in ligand specificity among members of mammalian odorant receptor subfamilies. In Aim 3, the Substituted Cysteine Accessibility Method will provide a rigorous test of our computationally derived OR models. In Aim 4, conventional mutagenesis and functional analysis, conducted based on sequence analysis of the subfamily members, will identify residues that confer differences in ligand specificity. This project will provide an understanding of ligand recognition by mammalian ORs at the molecular level. In addition to specific information about ligand recognition by ORs, this work will be applicable to GPCRs in general. This is important to human health because many current and potential drug targets are GPCRs. Our studies will provide fundamental information about ligand recognition by this important class of receptors. This information will be useful to future efforts in rational drug design. PUBLIC HEALTH RELEVANCE: Humans and other mammals detect odors using an enormous family of receptors (odorant receptors), which are similar in structure to many receptors that are current and future drug targets. This project examines the molecular basis for the ability of odorant receptors to distinguish among the many thousands of odor molecules. Information derived from this work will be useful in understanding the interaction between therapeutic drugs and their target receptors, and may aid in the design of more effective therapeutic drugs.

DESCRIPTION (provided by applicant): Many insect species exert a negative impact on human health through serving as disease propagation vectors or through causing agricultural damage. Chemosensory receptors, odorant receptors (ORs) and taste receptors, are critical to the ability of insects to function in these roles. Insect chemosensory receptors are an entirely novel receptor class, having no genetic or structural relationship to any known receptor family (including mammalian chemosensory receptors). Thus, these receptors are appealing targets for the development of new compounds for insect control. Realizing this goal requires development of a detailed understanding of insect chemosensory receptor structure and function. However, one of the most notable characteristics of these receptors is how little we know about their structure. In this project, we will identify structural features of insect ORs that are important in odorant binding and recognition. We have established a robust functional assay for insect odorant receptors using the Xenopus oocyte expression system and robotic electrophysiology. Using this assay, we are proposing a project that will determine the structural basis for odorant recognition by insect ORs. In Aim 1, we will express and screen a large panel of Drosophila melanogaster odorant receptors (DmORs) using our assay. In Aim 2, we will identify regions of interest on the odorant binding subunits of DmOrs by screening the receptor panel with cysteine-reactive methanethiosulfonate reagents. This screen will take advantage of the natural distribution of cysteine residues across the DmOR family. In Aim 3, we will use the Substituted Cysteine Accessibility Method (SCAM) to explore critical functional regions (identified in Aim 2) of the odorant binding subunits of insect ORs. In Aim 4, we will use site-directed mutagenesis and functional analysis to reveal the structural basis for odorant recognition by insect odorant receptors. This project will provide an understanding of odorant ligand recognition by insect ORs at the molecular level. This project is important to human health because insect ORs are a novel target for the development of compounds to control deleterious insect species. PUBLIC HEALTH RELEVANCE: Insects can have a negative impact on human health through serving as disease propagation vectors or through causing agricultural damage. Odorant receptors (ORs) are critical to the ability of insects to function in these roles. Insect ORs are an entirely novel receptor class, having no genetic or structural relationship to any known receptor family. In fact, insect ORs are not related to any proteins in humans and other mammals, making these receptors appealing targets for the development of compounds with higher selectivity and lower environmental toxicity than currently available insecticides. However, we currently know essentially nothing about the structure of these receptors. In this project, we will identify structural features of insect ORs that are important in odorant binding and recognition. This work will aid in the development of new compounds to control deleterious insect species.

Symbioses between eukaryotic hosts and bacterial consortia are fundamentally important to human health and ecosystem function. Gathering large amounts of DNA sequence data has become relatively easy and thus, models of host/microbiome function are accumulating. Critically, most models lack experimental tests. Working in a system that involves one eukaryotic host paired with a single bacterial symbiont the investigators will test models of symbiotic function to identify fundamental design principles in symbiosis.

The project will exploit a very productive collaboration to further examine the nature of nutrient transport and regulation in insects; specifically, the investigators will exploit the model aphid, Acyrthosiphon pisum, and its symbiont, Buchnera aphidicola, to fully characterize the substrate and feedback regulation of transporters between the aphid and bacterium. The first objective targets testing whole genome based models of symbiotic function and regulation. The research team will transiently express transporters of sap-feeding insects in oocytes of the South African clawed frog to test genome sequence based models of symbiotic function and host/symbiont regulation. Because aphids are both a significant crop pest and excellent models for the study of symbiosis any work that increases understanding of basic aphid biology increases our ability to develop ecologically safe methods of pest control and may facilitate development of the aphid as a model for understanding the process of bacterial infection. Functional characterization of transporters in the pea aphid and related insects will facilitate comparative work in other insect systems thus benefitting the annotation of transporters in all arthropod genomes. The second objective targets bringing NSF-funded basic research into the lives of Americans through the production of six broadcast quality films about aphids, the science of symbiosis and life as a scientist. The films will be supported by development of curricular resources that target early childhood and middle school children. Finally, this project supports the research program of a mid-career female scientist.