The ability to selectively manipulate the function of nicotinic acetylcholine receptors (nAChRs) will have wide ranging impact on the ability to understand the role of these receptors normal and pathological states. On the other hand, probes with which to characterize nAChRs are very limited. We have recently developed defining antagonists (alpha-conotoxins) for nAChRs. The project will exploit these recently developed probes to assess nAChR function and structure (Aims 1 &2). In separate, but complementary aims we will develop new antagonists for critical nAChR subtypes for which there are no selective ligands. Specifically we propose to:
Aim 1) Test the hypothesis that blocking alpha9alpha10 nAChRs is mechanistically responsible for analgesia and/or reduction of immune cell accumulation associated with nerve injury. Verification would establish a novel approach for the treatment of nerve injury and associated pain. We have developed a panel of subtype selective alpha-conotoxins that will be used together with receptor subunit knockout mice to achieve this aim.
Aim 2) Use toxin-receptor mutagenesis studies to elucidate the critical toxin/receptor interactions that underlie subtype selectivity. nAChR subtypes containing closely related alpha6- and alpha4-subunits presynaptically modulate the release of dopamine and norepinephrine that are implicated in addiction, Parkinson's disease and mood disorders. Existing small molecules cannot distinguish well among these subunits. In contrast, an existing set of alpha-conotoxins (different than those in """"""""Aim 1"""""""" above) are the most selective ligands known. We will use these toxins to provide mechanistic insight into selective action at alpha6- vs. alpha4-containing subunit interfaces.
Aims 3 -4) Develop and characterize new antagonists of nAChRs from Conus. Currently, selective ligands are only available for a minority of known nAChR subtypes, severely limiting our understanding. We will develop antagonists of nAChR subtypes for which there are no selective antagonists;we will focus on alpha4betaalpha5, alpha3beta4alpha5, alpha6beta4 and alternate stoichiometries of alpha4beta nAChRs, subtypes believed important in addiction and neuropsychiatric disorders. Conus is the richest natural source of nAChR antagonists that have been refined through 50 million years of evolutionary pressure. Development of this cornucopia will be achieved through biochemical characterization of venom compounds (Aim 3) as well as a gene cloning and iterative synthesis approach (Aim 4). Hundreds of alpha-conotoxins genes have been cloned and the encoded peptides will be synthesized. These peptides will then be fully characterized with respect to receptor subtype selectivity. Subsequently, synthetic strategies will be utilized to develop second-generation ligands with refined selectivity. Development of these antagonists will enable us to functionally knock out the critical nAChR subtypes including those that contain alpha4, alpha5 and alpha6 subunits. This will not only provide us with a platform for further mechanistic insight into nAChR function, but also will provide the wider scientific community probes to meet vital, but currently unfulfilled, needs (see Letters of Support, Significance, Progress, and Resource Sharing).
1) We will use selective antagonists of nicotinic receptors (conotoxins) together with genetically altered mice to gain insight into a novel mechanism for treatment of nerve injury and associated pain. 2) We will conduct structure-function analysis to elucidate the basis of the unique ability of conotoxins to distinguish between nicotinic receptors crucial to nicotine addiction. 3) We will exploit a rich natural product resource (Conus) to develop new compounds to enable pharmacological knockout of receptor subtypes for which there are no current ligands and which are mechanistically important in neurocognitive disorders.
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