STRUCTURE AND FUNCTION OF GLUTAMATE RECEPTORS

描述：拟议的实验旨在确定以下因素的作用： 多胺对某些类型谷氨酸的整流作用 受体。 调查员提出了三个具体目标， 谷氨酸受体功能和多胺作用的一般问题。 的 第一个目标是确定特定的多胺在赋予 观察到特定类型谷氨酸盐的向内整流 受体。 为此，调查人员将阻止生产 非洲爪蟾卵母细胞和哺乳动物细胞中的特定类型的多胺， 已经被编码谷氨酸受体的RNA转染。 块 将通过多胺水平的HPLC分析来证实， 功能性后果将通过测量谷氨酸 诱发了外向电流 这些实验提供的信息 将证明，多胺是内源性来源的整改 以及鉴定负责的特定多胺类型。 在第二 一组实验，研究人员将确定的灵敏度 不同的谷氨酸受体类型和突变型受体类型， 多胺 初步证据表明，GluR 4具有更高的 对精胺的敏感性高于GluR 1，尽管假定的 孔形成区（TM 2）在这两个亚基之间是相同的。 因此，研究者将使用这些信息 设计GluR 1和GluR 4嵌合体以定位额外孔的实验 贡献区域。 在最后一组实验中，研究人员将 寻找现行整改的依据， 谷氨酸受体。 在具体目标2中，研究者将确定 负责AMPA和红藻氨酸盐渗透性差异的残基 钙离子受体通道。 调查人员建议，根据 与电压依赖性通道同源，在TM 2外的孔衬环。 将在GluR 3和GluR 6受体亚基上进行点突变， 显示影响电压依赖性或配体门控的性质的位置 渠道 对二价阳离子的渗透性和对 对每种突变评估多胺。 在具体目标3中， 研究者将对野生型和突变型进行单通道研究 谷氨酸受体，以确定基础的外向 整改 三个不同的潜在整改来源将是 直接测试;在单通道级进行整流， 电压依赖性门控和多个电导， 电压依赖的。 从这些目标中获得的信息呈现出 考虑到最近披露的谷氨酸盐 三级结构似乎不同于配体门控的其他成员 受体家族

DESCRIPTION: The proposed experiments are aimed at establishing the role of polyamines in conferring rectification to certain types of glutamate receptors. The investigator presents three specific aims relating to the general problem of glutamate receptor function and polyamine action. The first aim will determine the role of specific polyamines in conferring the observed inward rectification observed for specific types of glutamate receptors. For this purpose the investigator will block the production of specific types of polyamines in Xenopus oocytes and mammalian cells which have been transfected with RNA coding for glutamate receptors. The block will be confirmed by HPLC analysis of the polyamine levels and the functional consequences will be determined by measurement of glutamate evoked outward currents. The information provided from these experiments will demonstrate that polyamines are the endogenous source of rectification as well as identifying the specific polyamine type responsible. In a second set of experiments the investigator will determine the sensitivity of different glutamate receptor types and mutant receptor types to different polyamines. Preliminary evidence suggests that GluR4 has a higher sensitivity to spermine than GluR1 in spite of the fact that the putative pore forming regions (TM2) are identical between these two subunits. Therefore, the investigator will use the information obtained from these experiments to design GluR1 and GluR4 chimeras to locate additional pore contributing regions. In a final set of experiments the investigator will search for the bases of current rectification which is observed for Glutamate receptors. In Specific Aim 2 the investigator will determine the residues responsible for differences in permeability of AMPA and Kainate receptor channels to calcium. The investigator proposes, on the bases of homology to voltage-dependent channels, a pore lining loop outside TM2. Point mutations will be made on GluR3 and GluR6 receptor subunits at locations shown to affect properties of voltage-dependent or ligand gated channels. The permeability to divalent cations and the sensitivity to polyamines will be assessed for each mutation. In Specific Aim 3 the investigator will perform single channel studies on wild type and mutant glutamate receptors in order to ascertain the bases for outward rectification. Three different potential sources of rectification will be directly tested; rectification at the single channel level, voltage-dependent gating, and multiple conductances which are voltage-dependent. The information obtained from these aims takes on additional significance given the recent disclosure that the Glutamate tertiary structure appears distinct from other members of the ligand gated receptor family.