ETHANOL SENSITIVITY OF NATIVE AND CLONED NMDA RECEPTORS

乙醇对中枢神经系统的作用广泛而多样 系统，表现在正常神经化学物质的改变 过程和行为。的细胞和分子作用部位 过去，中枢神经系统中的乙醇一直是集中研究的焦点 10年许多不同的神经过程已经被证明是 对急性和慢性暴露于乙醇敏感。其中，大 神经递质门控离子通道家族已被证明 对乙醇特别敏感。可以假设， 乙醇对中枢神经系统功能和行为的作用是由于选择性 乙醇对这些离子通道蛋白的影响。研究在我 实验室和其他人的研究表明，谷氨酸的一种亚型 受体，N-甲基-D-天冬氨酸受体，被乙醇抑制， 浓度是行为相关的过程中， 在目前的资助期间，我的实验室专注于确定 分子决定因素，赋予乙醇敏感性后，这些 受体。使用天然的和重组的NMDA受体， 脑神经元和异源表达系统，我们已经表明， 乙醇对NMDA受体抑制作用受亚基 与C-末端相互作用的组成和细胞内过程 NMDA受体亚单位的表达。我们最新的数据显示， 钙敏感组分和对乙醇敏感的不敏感组分 NMDA受体。钙敏感型涉及的C-末端 NR 1亚基具有不同的蛋白质结合位点， 磷酸化和蛋白质-蛋白质相互作用。这些过程 调节NMDA受体的活性，似乎是选择性的， 被乙醇改变。乙醇的钙不敏感成分 假设NMDA受体的抑制是通过 乙醇与构成跨膜的关键残基的相互作用 受体的结构域。在目前资助的R 01的延续中， 我们建议精确地定义分子位点和机制， 钙敏感和不敏感形式的基础作用 乙醇抑制NMDA受体。本研究将利用两个- 电极电压钳、单细胞钙成像和全细胞 膜片钳技术测定乙醇对NMDA受体的影响 在卵母细胞、HEK 293细胞和培养的神经元中表达。两 分子和药理学方法将被用来解开 受体和细胞内信号传导之间的复杂相互作用 最终决定NMDA对乙醇敏感性的过程 受体。

Ethanol causes widespread and varied actions on the central nervous system that are manifested in alterations in normal neurochemical processes and behavior. The cellular and molecular sites of action of ethanol in the CNS have been the focus of intense research over the past 10 years. Many different neuronal processes have been shown to be sensitive to acute and chronic exposure to ethanol. Of these, the large family of neurotransmitter-gated ion channels has been shown to particularly sensitive to ethanol. It can be hypothesized that many of the actions of ethanol on CNS function and behavior are due to selective effects of ethanol on these ion channel proteins. Research in my laboratory and that of others has shown that a subtype of glutamate receptor, the N-methyl-D-aspartate receptor, is inhibited by ethanol at concentrations at are behaviorally relevant During the course of the current funding period, my laboratory has focused on determining the molecular determinants that confer ethanol sensitivity upon these receptors. Using both native and recombinant NMDA receptors expressed in brain neurons and heterologous expression systems, we have shown that the ethanol inhibition of NMDA receptors is modulate both by subunit composition and intracellular processes that interact with the C-termini of NMDA receptor subunits. Our most recent data show that there are calcium-sensitive and insensitive components to the ethanol sensitivity of NMDA receptors. The calcium-sensitive form involves the C-terminus of the NR1 subunit which possesses distinct binding sites for protein phosphorylation and protein-protein interactions. These processes regulate the activity of the NMDA receptor and appear to be selectively altered by ethanol. The calcium-insensitive component of ethanol's inhibition of NMDA receptors is hypothesized to be mediated via an interaction of ethanol with key residues that make up the transmembrane domains of the receptor. In this continuation of a currently funded R01, we propose to precisely define the molecular sites and mechanisms of action that underlie the calcium-sensitive and insensitive forms of ethanol inhibition of NMDA receptors. This research will utilize two- electrode voltage clamp, single-cell calcium imaging and whole-cell patch clamp to measure the effects of ethanol on NMDA receptors expressed in oocytes, HEK 293 cells, and cultured neurons. Both molecular and pharmacological approaches will be used to unravel the complex interplay between receptors and intracellular signaling processes that ultimately determine the ethanol sensitivity of NMDA receptors.