ALLOSTERIC POTENTIATION OF NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 埃德蒙兹博士在2006-2007年间通过外展核心加入了INBRE支持学院。他已经成功地招募了一名研究生，这名研究生正式是费尔班克斯校区的博士生，但现在他在朱诺校区的埃德蒙兹博士实验室工作。 埃德蒙兹博士提出的明确的研究建议。 目的1：鉴定选择性变构增强剂去甲酰氟溴与乙酰胆碱受体a4b2亚型的结合部位。该受体胞外区的同源模建将用于确定dFBR的候选结合位点。结合位点(S)的鉴定将通过检查假定结合位点附近残基的系统性突变的功能效应来确认。 目的2：鉴定对变构位点的结合和活性起关键作用的去甲酰氟溴的分子特征。这一目标将使用标准的电生理学方法来筛选自然产生的dfbr的合成类似物。增强ACh作用的化合物将在目标3中进一步研究。活性化合物的结构和化学特征将被记录下来，并在设计过程中用于改善化合物的功能性质(S)。 目的3：确定研究目标2中确定的去甲酰氟溴类似物的增强机制。为此，我们将利用膜片钳方法结合快速激动剂应用系统来检测dFBR类似物(S)(目标2)对单个受体通道对ACh脉冲反应的影响。A4b2受体将从卵母细胞(瞬时表达)或稳定表达的HEK细胞中获得。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for 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.