Item No. 1 of 1

INVESTIGATOR: Leary, G. P.

PERFORMING INSTITUTION:
UNIVERSITY OF MONTANA
COLLEGE OF FORESTRY AND CONSERVATION
MISSOULA, MONTANA 59812

STRUCTURE AND FUNCTION OF THE EUROPEAN CORN BORER SEX PHEROMONE RECEPTORS

NON-TECHNICAL SUMMARY: The insect order Lepidoptera (moths and butterflies) includes over 180,000 species and is one of the largest lineages of plant eating organisms (Grimaldi & Engel, 2006). Reproduction in moths almost exclusively depends on a unique pheromone blend released from the female's pheromone gland which is detected by highly specific odorant receptors of the male antenna over distances of 10 to 1000 meters. This ability to locate mates at long distances has allowed for the extensive spread of moth species, and it also provides an ideal target for the management of species, specific, pest populations. The potential for adopting bio-rationale approaches to developing semiochemicals for insects has surged with the identification of a large number of olfactory receptors genes (Clyne et al, 1999, Vosshall et al, 1999, Gao and Chess,1999). In this proposal, we use the male specific European Corn Borer (ECB) sex pheromone receptors (ECBOR 1 and 3-7 expressed with the co-receptor ECBOR2) as a model system to distinguish these homologous receptors (Wanner et al, 2010). With an eventual goal to rationally design molecules for the Lepidoptera sex pheromone receptors and insect ORs in general, we utilize methods pioneered in pharmaceutical drug design that is rooted in heterologous expression systems, a basic understanding of receptor-ligand interaction, and a fundamental knowledge of the protein signal transduction mechanism.

OBJECTIVES: In Objective 1, we will develop a large data set defining the molecular pharmacology for each ECBOR (1 & 3-7). In the Lepidoptera, most mating pheromones are long hydrocarbon chains that have variations of carbon chain length, type of terminal functional group, double bond location, number of double bonds, and/or stereochemistry around the double bond. From these five characteristics, we have created a library of European corn borer pheromone related molecules to test on ECBOR1 & 3-7 expressed in Xenopus laevis oocytes with the necessary co-receptor ECBOR2. This effort will provide a framework to identify the ligand structural determinants that confer receptor selectivity and agonist/antagonist activity in this receptor family. In Objective 2, we will identify the structural determinants in two closely related receptors, ECBOR3 and ECBOR6, that confer selectivity for ECBOR6 selectively binding of the primary mating pheromone, Z11-tetradecenyl acetate. Sequence alignment of the predicted transmembrane and extracellular domains reveals that ECBOR6 and ECBOR3 are 72.4% identical and 94% similar. The most obvious divergence in sequence is between the beginning of transmembrane domain (TMD) 3 and the end of TMD4, which includes the largest extracellular domain of the protein. We hypothesize that residues within these transmembrane and extracellular domains form direct amino acid contacts that distinguish carbon chain length and stereochemistry of the pheromones. We will test this by engineering chimeras and using site directed mutagenesis to define changes that convert the highly specific response profile of ECBOR6 to the broad response profile of ECBOR3. In Objective 3, we will characterize the the novel pheromone-gated ion channel in this seven transmembrane domain pheromone receptor family. My preliminary data show that application of the thiol reactive probe, methane thiosulfonate (MTSEA), selectively blocks a conductance in oocytes expressing ECBOR3 and the mandatory co-receptor ECBOR2. Two separate research groups reported a cation channel within the insect odorant receptors that is independent of Gprotein activation; however, the activating mechanism and location of the channel remains unresolved (Sato et al, 2008 and Wicher et al, 2008). We hypothesize that MTSEA modifies an endogenous cysteine residue in the receptor that either blocks the permeation pathway for cations or directly interferes with gating of the channel. Through site directed mutagenesis, we will identify which of seven extracellular cysteine residues is modified and accounts for the block. We will distinguish the effects in ion permeation vs ion gating by studying how MTSEA affects the single channel currents recorded in outside-out patches pulled from ECBOR3&ECBOR2 expressing oocytes. Finally, we will define the channel structure using cysteine-scanning mutagenesis.

APPROACH: Two electrode voltage clamp. European corn borer sex pheromone receptor cRNA will be microinjected in stage V-VI Xenopus oocytes. Equal amounts of ECBOR2 and ECBOR (1, 3, 4, 5, 6, or 7) linearized DNA will be combined and then transcribed to cRNA using mMessage Machine Kit (Ambion). Recording solution (frog Ringer) will contain 96mM NaCl, 2mM KCl, 1mM MgCl2, 1.8mM CaCl2, and 5mM HEPES (pH7.4). Microelectrodes will be pulled to a resistance between 1 MΩ and 3 MΩ and filled with 3M KCl. Data will be recorded with power lab 2/20 (AD instruments) interfaced with a Macintosh computer using Chart v7.1.2 software. Data will be analyzed offline with Axograph X (v1.0.8) and Kaleidagraph (v3.6). We will use methods similar to methods used in Leary et al., 2007. Outside-out patch clamp recordings- ECBOR3+ECBOR2 will be microinjected in stage V-VI oocytes. Oocytes will be check for expression on days 3-5 before pulling patches from the oocyte membrane. After removal of the vitelline membrane, outsideout patch recordings will be made with fire-polished pipettes (4-7 MΩ) as previously described (Wadiche and Kavanaugh, 1998). The extracellular solutions used will be 96mM NaCl, 2mM KCl, 1mM MgCl2, 1.8mM CaCl2, and 5mM HEPES (pH7.4) . The intracellular recording solution will be 110mM KCl, 1mM MgCl2, 10mM EGTA, 5mM HEPES (pH7.4 with TRISbase). The outside-out patches will be held at -80 mV as in Figure 3. Membrane currents will be recorded with a Multiclamp 700B interfaced to Macintosh computers with Digidata 1430 A/ D interfaces. Only patches with membrane seal resistances > 5 GΩ will used in experiments from either uninjected or ECBOR injected oocytes. Records will be low-pass filtered at 1 kHz and digitized at 2kHz.

PROGRESS: 2011/08 TO 2014/08
Target Audience: The second year of my work has aimed to extend my current research on insect olfactory receptors and expand my future research to include insect gustatory receptors. I have pursued my continual participation in establishing a collaboration between Dr. Kevin Wanner at Montana State and Dr. Mike Kavanaugh at the University of Montana. With Dr. Kevin Wanner, I have aided in writing and submitting a USDA grant that targets the role of pheromone binding proteins within the primary response to pheromones and there relationship to sex-pheromone receptors in moths. With Dr. Michael Kavanaugh, I have maintained an active role as a prominent researcher within his lab that both facilitates undergraduate and graduate researchers as well as lectures on specific neuroscience related topics. I have begun collaborations with Dr. Sarah Certel at the University of Montana to begin using fluorescent calcium imaging in Drosophila lines to measure gustatory responses to pheromones throughout specific gustatory neurons. This collaboration is designed to develop a “empty gustatory neuron” system in Drosophila that is similar to the one currently used for olfactory receptors. Because minimal progress has been made on expressing insect Grs in isolation, implementing them into a viable gustatory neuron should incorporate the necessary chaperone proteins to allow for functional analysis of individual gustatory receptors from any species. A second audience that I targeted for sharing my current research was with Dr. Walter Leal and his laboratory at UC Davis. Dr. Leal is a renowned scientist in studying the molecular mechanisms of insect olfaction and gustation. He has a long history in the study of pheromone binding protein that make up much of the extracellular space surrounding insect Ors and Grs and is well established in the application of these principals to agriculture. Finally, I have extended my research toward developing an insect cell expression system by beginning work with macromolecular X-ray diffraction core facility at the University of Montana, which is run by Dr. Steve Sprang. The goal in targeting this audience is to purify recombinant odorant and gustatory receptors protein from the insect chemoreceptor superfamily within an established protocol that can lead to X-ray crystal structure of a protein of this family. In scientific fields where a new membrane protein has been crystallized, the findings have often opened new avenues for applying computational drug design to guide structure/function hypotheses and small molecule development. Crystallizing an insect olfactory receptor will provide a 3-dimensional structural model that will serve as a framework for other related members of the insect chemosensory receptor superfamily. This will fill a fundamental gap in our knowledge and understanding of how these receptors allow insects to detect chemicals in their environment and translate this signal into a biologically relevant response. The results will have the potential to play a critical role in rational design and development of compounds that can interfere with receptor signaling while minimizing ecological damages. Furthermore, our research will establish the necessary experimental conditions and procedures to crystallize critical ORs and GRs from other insects that mediate crop damage or serve as vectors for human diseases. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Through the second year of my work, I focused on both experimentation and searching for work following my post doctoral fellowship. For experimentation/training opportunities, I spent time traveling between Bozeman and Missoula working between the two labs. Both experiences allowed me to access techniques unique to molecular biology and electrophysiology as well as share these information sources between the two labs. In terms of searching for a new job, the grant gave me both funding and leverage to apply for positions in the field of entomology. I used the grant to travel to Davis California to interview for a position in Dr. Walter Leal’s lab. I also have pursued potential research position at the Notre Dame, University of Montana, and Montana State in Bozeman. How have the results been disseminated to communities of interest? Our main outreach, opposed to specfic individuals within our local scientific community that I highlighted in our tagret audience, was to present our results at the ESA annual meeting (Entomological society of America). In addtion, we have a local neuroscience meeting that collects neuroscience researcher from around the state of Montana that was held in Bozeman that we reported our work. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2011/08 TO 2014/08
What was accomplished under these goals? Although insects have a large numbers of olfactory (ORs) and gustatory receptors (GRs) tuned for different purposes (30-200 receptor genes depending on the insect species), all of the receptors share a common structural backbone of the protein and thus a common mold across all insects that is essential for the olfactory and gustatory receptor function. Many basic questions remain about the receptors in the insect chemosensory receptor superfamily: 1) What is the multimeric nature of the receptor complex? 2) Where are the critical binding domains within the protein? 3) How are these binding domains altered for different receptors? 4) How does the binding of pheromone translate to the opening of the existing ion channel? 5) Are there unique protein binding domains that can be chemically targeted only when the ion channel is activated? 6) What are the structural similarities and difference between insect olfactory and gustatory receptors? My work begins with a basic science approach to guide the development of species-specific solutions to insect depredation by targeting the odorant and gustatory receptors that underly life sustaining behaviors such as mating, locating food resources, and avoiding danger. Although my work specifically focuses on moth sex pheromone receptors (one type of odorant receptor), the biophysical and structural questions I am asking have many broad applications to all species of insects. Throughout my work in this second year, I tried to research my proposed aims as well as venture into a series of techniques that would allow me to ask more advanced questions about the biophysical properties of insect olfactory and gustatory receptors. As proposed in my research, I used the Xenopus oocyte system with two-electode voltage clamp to study electrogenic currents associated with the expression of moth sex pheromone receptors. In these efforts I focused on screening a series of pheromones targeted toward corn borer Ors (asian and european corn borer moths) in addition to ligands that possibly could target an agonist binding site on the obligate coreceptor ORCO. To diversify this pharmacology screening approach in addition to develop purified protein for potential crystallization efforts, I cloned and expressed a couple corn borer sex-pheromone receptors and ORCO into an insect cell expression system (Sf9 cells). Using calcium sensitive dyes, I was able to measure fluorescent changes in response to ORCO agonists and pheromones verifying the functional expression of these proteins. Using a cell line as opposed to oocytes, offers larger screening potential in 96 well plates. My biophysical research combined TEVC recordings with single channel patch clamp recordings and focused on three hypotheses related to how different environmental conditions affect the odorant receptor function: 1) The unique lymph solution surrounding the olfactory neuron dendrites (200mM potassium, low pH, with a high reducing potential) has a significant impact on that functional response of the moth sex pheromone receptor; 2) Though calcium is permeable to insect ORs, a high affinity calcium binding site blocks the channel at physiological concentrations (2mm) and maintains the excitability of the membrane at rest. 3) Insect odorant receptors are ligand-gated ion channels that function independent of G-protein coupled receptors (GPCRs), still a controversial perspective. In each of these areas, I have found that environmental components surrounding the ORs significantly alter the receptor kinetics. Separately, I have begun to clone insect gustatory receptors in oocyte expression vectors to see if I can functionally record electrogenic responses. Biophysically, the oocyte expression system is a robust and convenient way to characterize a novel receptor or transporter. Initially, I have expressed a conserved fructose receptor from drosophila, Gr43a, and shown a clear response to fructose in ococytes. This compliments work published previously on a silk worm fructose receptor (Sato et al., 2011). In addition, I have tried to express a sex pheromone gustatory receptor from Drosophila, Gr32a, although I have not been able to confirm a functional response in oocytes. For this reason, I have paralleled the gustatory recordings with recordings using genetically driven fluorescent reporters in drosophila gustatory neurons that express the Gr32a receptor. We have developed a recording assay that shows a clear fluorescent resposne within the living fly for bitter compounds in these neurons, but have not confirmed the agonist that targets Gr32a. I currently have no publications from this year of the grant, though I am working on writing up a number of these final results. I will also use much of my current data as preliminary data for future grants as I begin searching for a permanent faculty position. One grant that I helped right with Dr. Kevin Wanner was just funded, which I feel is a testimant to the quality of research we are working towards as well as our 2012 PNAS publication on research proposed in my post doctoral grant. I feel this post doctoral grant has offered me the freedom to pursue scientific avenues that I believe will soon substatially contribute to the managment of insect pests within both agricultural and health related communties.

PUBLICATIONS (not previously reported): 2011/08 TO 2014/08
No publications reported this period.

PROGRESS: 2011/08/15 TO 2012/08/14
OUTPUTS: Activities The ability to produce innovative and world class research in Montana can most effectively be accomplished by institutions around the state collaborating through ideas and sharing of resources/equipment. Although many world renowned scientist choose to relocate to Montana, one barrier between academic/research institutions is often the more than 200 miles that separates major cities. Receiving this USDA post doctoral fellowship has allowed me to stabilize a collaboration between biophysical research performed at the in Dr. Mike Kavanaugh's Lab at the University of Montana, Missoula, and research on the genetic cloning on insects chemoreceptors in Dr. Kevin Wanner's lab at Montana State University, Bozeman. At Montana State University, I have been essential in establishing electrophysiology recordings from Xenopus oocyte that express a variety of insect chemoreceptors that Dr. Wanner's lab specializes in cloning from insects. In these tasks, I have played a critical role in instrument setup, configuration, software interface, and experimental recording. In addition, I have shared procedures for surgical removal of oocytes from Xenopus lavies frogs and processing to produce viable recording samples. At the moment, Dr. Wanner and his technician can perform all the necessary steps to acquire functional responses from insect ORs. In addition, I have contributed a role in interpreting and analyzing results from the recordings. These are not trivial analysis and are rooted in basic protein-receptor biophysics. At the University of Montana, I have been pioneering single channel recording from the oocytes expressing insect chemoreceptors. This method will add to the complexity in which we can analyze the insect chemoreceptors and begin to establish an isolated system to understand how multiple components within the antenna of insects directly effect the insect olfactory receptor. In combination with this research, I have established an essential role as mentor to both undergraduate and graduate students as well as a temporary lecturer of an undergraduate Neuroscience course. Events Though the first year of my work has focused mainly on experimentation, I have presented our work at both the NIFA fellowship meeting in Washington DC as well as a state conference that focuses on collaborative research between the private and public research institutions in Montana. In addition, I spent time traveling between Bozeman and Missoula working between the two labs. Products I am currently collaborating with the Department of Mathematics at the University of Montana to develop an open source data acquisition and analysis software for electrophysiology and imaging equipment. At the moment I are using a functional version of the software for all my experimentation, and working on developing cutting edge Applied Mathematical modeling tools to analyze complex Markov Chain models related to insect chemoreceptors. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

IMPACT: 2011/08/15 TO 2012/08/14
The aim of my proposal focuses on understanding the structure and functional aspects that allow insect olfactory receptors to detect unique pheromones in closely related species of moths. These receptors belong to a novel family of proteins that include insect gustatory and olfactory receptors. A number of fundamental questions about how these receptors work remain which require an answer before these proteins can effectively be used as targets to alter insect behavior such as reproduction. In our first publication, we took advantage of closely related moth species, the european corn borer and the asian corn borer, and compared the functional response of a select number of sex pheromone receptors unique to the male antenna. These sex pheromone receptors distinguish the mating pheromones between the two species, two pheromones that only differ by a subtle shift in a carbon double bond position. Since only a few mutations differentiate these essential receptors from the two species, we were able to identify an essential binding region in these receptors in the third transmembrane domain. We also were able to mutate the receptor to change the functionality of the european corn borer to that of the asian corn borer, demonstrating our ability to alter the pheromone binding region.

PUBLICATIONS: 2011/08/15 TO 2012/08/14
1. Leary GP et al. (2012) Single mutation to a sex pheromone receptor provides adaptive specificity between closely related moth species. Proc. Natl. Acad. Sci. U.S.A. 109:14081.
2. Rau TF et al. (2012) Oxygen glucose deprivation in rat hippocampal slice cultures results in alterations in carnitine homeostasis and mitochondrial dysfunction. PLoS ONE 7:e40881.