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Curriculum Vitae
Research InterestsThe research in my laboratory is directed toward understanding the structural basis for receptor function. We have two main areas of focus: 1) Nicotinic acetylcholine receptors (nAChRs) expressed in the mammalian nervous system; 2) Mammalian odorant receptors. Neuronal Nicotinic ReceptorsThese receptors are of interest for several reasons. Nicotinic ligands are potentially useful in the treatment of dementias, and as anxiolytics and analgesics. Neuronal nAChRs are also the sites at which nicotine exerts its psychoactive and addictive effects. Thus pharmacologic intervention at neuronal nAChRs holds promise for treating the effects of diseases of the central nervous system and for understanding and treating addictive processes. Critical to the realization of this potential, is the development of an understanding of the molecular basis for pharmacological diversity among neuronal nicotinic receptor subtypes. Project 1) Modulation of neuronal nicotinic receptors by zincIonic zinc has been found in neurons throughout the brain, with highest concentrations in the cerebral cortex and limbic areas. Zinc is localized to small, clear vesicles in synaptic terminals and is released in a calcium dependent manner. During neuronal activity, the extracellular concentration of zinc is estimated to reach concentrations as high as several hundred micromolar. We recently discovered that some, but not all, neuronal nAChR subtypes are potentiated by low zinc concentrations (1-100 µM), while all neuronal nAChR subtypes are inhibited by high zinc concentrations (> 100 µM). The extent of potentiation by zinc varies widely depending on receptor subunit composition. Potentiation by zinc represents a new mode of neuronal nAChR modulation. We are currently working to identify the amino acid residues involved in binding zinc at the potentiating and inhibitory sites. We are also examining the mechanism by which zinc potentiates neuronal nAChR function by examining the effects of zinc at the single channel level (in collaboration with Dr. Karl Magleby, Department of Physiology and Biophysics). Project 2) The contribution of alpha and beta subunits to the pharmacological properties of neuronal nAChRsNeuronal nicotinic receptors, like muscle nicotinic receptors, are pentameric assemblies of various combinations of subunits. Neuronal nicotinic receptor subunits are designated alpha and beta, with many distinct forms of each being present in the nervous system. Both the alpha and beta subunits make contributions to the pharmacological properties of these receptors. We are working to identify and understand the contribution of each class of subunit to the pharmacological properties of these receptors. Previously characterized pharmacological differences among distinct subunit combinations provide the starting point for this work. Initially we constructed a series of chimeric subunits, composed of portions of pharmacologically differing subunits. Analysis of receptors formed by these chimeras in Xenopus oocytes allowed identification of protein segments that conferred pharmacological differences. Within these identified regions, we then proceeded to change individual amino acid residues, from the naturally occurring residue, to what occurs in a pharmacologically distinct subunit. Analysis of receptors formed by these mutant subunits has allowed identification of a number of residues, on both alpha and beta subunits, that confer pharmacological specificity. Currently, the laboratory is working to understand the role of these identified residues by systematically varying residue side chain characteristics. In a collaboration with Dr. Michael McIntosh (University of Utah), we are exploring the interaction between alpha-conotoxin-MII (a 16-residue polypeptide antagonist of known three-dimensional structure) and neuronal nAChRs. We are examining the interaction between mutant conotoxins and mutant neuronal nAChRs. By comparing this data with the three-dimensional structure of alpha-conotoxin-MII and a homology modeled structure of the extracellular domain of neuronal nAChRs (developed in collaboration with Dr. Arun Malhotra, Department of Biochemistry and Molecular Biology), we will determine the structural basis for the interaction between a neuronal nAChR and a ligand. Project 3) SAR of EpibatidineIn collaboration with Dr. Ivy Carroll (Research Triangle Institute) we are determining the effects of modifications to the structure of epibatidine (an exceptionally potent nicotinic agonist). A series of epibatidine analogs have been and are being produced by Dr. Carroll's laboratory. We are using electrophysiological and radioligand binding techniques to determine the neuronal nicotinic subtype selectivity (in terms of affinity, functional potency and efficacy) of these analogs. This work will lead to a more detailed SAR (Structure-Activity Relationship) for agonists acting on neuronal nAChRs. Mammalian Odorant ReceptorsMammals are capable of distinguishing among thousands of odorants and the first step in this process is the interaction between odorant ligand and olfactory 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 have suggested that members of an OR subfamily recognize similar chemical structures. We are trying to understand 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. We are using this screening system to identify ligands for mouse and human ORs. As we identify ligands for particular ORs, we are cloning and functionally express other members of the OR subfamily. We then compare the ligand specificities of members of OR subfamilies. Detailed pharmacological analysis is conducted to reveal differences in ligand specificity among subfamily members. We will then work to identify the structural basis for differences in ligand specificity of members of mammalian odorant receptor subfamilies. Site-directed mutagenesis experiments, conducted based on sequence analysis of the subfamily members, will identify residues that confer differences in ligand specificity. Computational homology modeling and ligand docking will provide predictions that can be tested in further mutagenesis experiments. Recent PublicationsHsiao, B., Mihalak, K.B., Magleby, K.L., Luetje, C.W. (2008) Zinc
potentiates neuronal nicotinic receptors by increasing burst duration.
Journal of Neurophysiology, in press. Abaffy, T., Malhotra, A., Luetje, C.W. (2007) The molecular basis for ligand specificity in a mouse olfactory receptor: A network of functionally important residues. Journal of Biological Chemistry 282: 1216-1224. Shiembob, D.L., Roberts, R.L., Luetje, C.W., McIntosh, J.M. (2006) Determinants of alpha-conotoxin BuIA selectivity on the nicotinic acetylcholine receptor beta subunit. Biochemistry, 45:11200-11207. Mihalak, K.B., Carroll, F.I., Luetje, C.W. (2006) Varenicline is a partial agonist at alpha4beta2 and a full agonist at alpha7 neuronal nicotinic receptors. Molecular Pharmacology, 70:801-805. Abaffy, T., Matsunami, H., Luetje, C.W. (2006) Functional analysis of a mammalian odorant receptor subfamily. Journal of Neurochemistry 97: 1506-1518. Hsiao, B., Mihalak, K.B., Repicky, S.E., Everhart, D., Mederos, A., Malhotra, A., Luetje, C.W. (2006) Determinants of zinc potentiation on the alpha4 subunit of neuronal nicotinic receptors. Molecular Pharmacology 69: 27-36.
Current Lab Members
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