This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Dr. Edmonds joined the INBRE support faculty through the outreach core in 2006-2007. He has successfully recruited a graduate student that is officially a doctoral student of the Fairbanks campus however is working now in the Dr Edmonds lab on the Juneau campus.Defined research proposal from Dr. Edmonds.
Aim 1 : Identify the binding site of the selective allosteric potentiator deformylflustrabromine on the a4b2 subtype of the acetylcholine receptor. Homology modeling of the extracellular domain of the receptor will be used to identify candidate binding sites for dFBr. Identification of the binding site(s) will be confirmed by examining the functional effects of systematic mutagenesis of residues in the vicinity of the putative binding site.
Aim 2 : Identify the molecular features of deformylflustrabromine critical for binding and activity at the allosteric site.
This aim will be addressed using standard electrophysiological methods to screen synthetic analogs of naturally occurring dFBr. Compounds that potentiate the action of ACh will be investigated further in Aim 3. The structural and chemical features of active compounds will be noted and used in the design process to improve functional properties of the compound(s).
Aim 3 : Determine the mechanism of potentiation for the deformylflustrabromine analogs identified in Research Aim 2. For this aim we will utilize patch clamp methodology in combination with a fast agonist application system to examine the effects of dFBr analog(s) (Aim 2) on single receptor-channel responses to pulses of ACh. a4b2 receptors will be harvested either from oocytes (transient expression) or stably transfected HEK cells.
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