STRUCTURE AND FUNCTION OF GLUTAMATE RECEPTORS

描述：拟议的实验旨在确定 多胺对某些类型谷氨酸的整流作用 感受器。调查员提出了三个具体目标，与 谷氨酸受体功能和多胺作用的一般问题。这个 第一个目标将确定特定多胺在赋予 观察到特定类型的谷氨酸的内向整流 感受器。为此目的，调查员将阻止生产 非洲爪哇卵母细胞和哺乳动物细胞中特定类型的多胺 已经被导入了编码谷氨酸受体的RNA。这座街区 将通过对多胺水平的高效液相分析和 功能后果将通过谷氨酸的测量来确定 诱发了向外的电流。从这些实验中提供的信息 将证明多胺是精馏的内源性来源 以及确定具体的多胺类型负责。一秒钟内 研究人员将确定的一系列实验的敏感性 不同谷氨酸受体类型和突变受体类型对不同 多胺。初步证据表明，GluR4具有更高的 对精胺的敏感性高于GluR1，尽管推测的 这两个亚基之间的孔隙形成区域(TM2)是相同的。 因此，调查员将使用从这些文件中获得的信息 设计GluR1和GluR4嵌合体以定位额外孔洞的实验 贡献地区。在最后一组实验中，调查员将 寻找当前整改的观察依据 谷氨酸受体。在具体目标2中，调查员将确定 影响AMPA和海人藻酸盐渗透性差异的残留物 钙离子的受体通道。调查员建议，在以下基础上 与电压依赖通道同源，在TM2外有一个孔衬环路。 GluR3和GluR6受体亚基将发生点突变 影响电压依赖性或配位体门控性质的位置 频道。对二价阳离子的渗透性和对 将对每个突变的多胺进行评估。在具体目标3中 调查员将对野生型和突变型进行单通道研究 谷氨酸受体，以确定向外的基础 整改。三个不同的潜在整改来源将是 直接测试；单通道级别的整改， 与电压相关的选通，以及多个电导 电压相关。从这些目标获得的信息具有 考虑到最近披露的谷氨酸 三级结构似乎与配位体门控的其他成员不同 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.