酒精敏感型结构域在慢性酒精性NMDA受体表达调节中的分子机制研究

NMDA receptors play a crucial role in the etiology and progression of alcohol-induced neurological and psychiatric disorders. Acute alcohol exposure inhibits NMDA receptor function, but chronic alcohol intake increases NMDA receptor expression and function. The mechanism of this regulation, however, is still unclear. Our previous studies have identified a number of positions in the membrane-associated (M) domains M3 & M4 of the GluN1 and GluN2A subunits that regulate acute alcohol action. Related sites are likely to be involved in regulation of chronic alcohol-induced increase of NMDA receptor expression. In light of these findings,we will use point mutations, western-blot, immunofluorescence, and electrophysiological patch clamp techniques, as well as models of chronic alcohol-treated primary neurons in vitro, to investigate the molecular mechanisms by which ethanol acts on these alcohol-sensitive domains to modulate NMDA receptor expression directly, and to determine whether key alcohol-sensitive positions might influence NMDA receptor function by altering subunit retention in the endoplasmic reticulum. The proposed studies represent the key basic research for development of new specific and efficient drugs to target NMDA receptor sites of alcohol action. The information obtained from these studies is expected to lead to the identification of novel target sites and the development of new strategies for the treatment of NMDA receptor derangement and its consequences in alcohol abuse and alcoholism.

NMDA受体在酒精性神经精神疾病的发病及病理进程中起着至关重要的作用。急性酒精刺激抑制NMDA受体功能，慢性酒精摄入则使NMDA受体表达增加功能上调，但此机理仍不清楚。申请人前期研究在NMDA受体的GluN1、GluN2A亚单位膜相关功能区M3、M4上鉴定出多个调控急性酒精作用的位点，相关位点很可能参与调控慢性酒精性NMDA受体的表达。基于此，本项目拟通过点突变、免疫印迹、免疫荧光、膜片钳以及原代神经细胞慢性酒精刺激模型,研究酒精如何通过这些酒精敏感型结构域直接调控NMDA受体表达，以及关键酒精敏感性位点是否通过影响NMDA受体亚单位内质网滞留进而调控NMDA受体功能的分子机制。本项目是一项针对NMDA受体酒精作用位点研发新型特效药物的关键前期基础研究，其研究成果有望为酒精滥用和中毒引起的NMDA受体功能紊乱及所致神经精神疾病提供全新的治疗靶点和治疗新思路。

NMDA受体在酒精性神经精神疾病的发病及病理进程中起着至关重要的作用。急性酒精刺激抑制NMDA受体的功能，慢性酒精摄入则使NMDA受体表达增加功能上调，但此机理仍不清楚。申请人前期研究在NMDA受体的GluN1、GluN2A亚单位的M3、M4上鉴定出多个调控急性酒精作用的位点，相关位点很可能参与调控慢性酒精性NMDA受体的表达。在此基础上，本项目重点研究M3、M4酒精敏感性结构域位点对NMDA受体表达的影响。发现：1. 酒精敏感性位点GluN2A(F636)不仅调节急性酒精的敏感性，还影响细胞表面单位面积NMDA受体的数量。2. 不同的氨基酸替代酒精敏感性位点GluN2A(F637)的氨基酸残基，对细胞表面单位面积受体数量的影响存在显著性差异。3.不同的酒精敏感性位点，在调节NMDA受体亚单位总蛋白的表达上亦存在差异。 4.位于GluN1和GluN2A上第三或者第四跨膜区上的等位点的酒精敏感性氨基酸残基位点，在调节酒精敏感性及调节受体表达的作用上并非相同。结果表明：NMDA 受体酒精敏感性结构域不仅存在影响 NMDA 受体酒精敏感性和离子通道功能的位点，而且关键位点对受体的表达亦起着重要的调节作用。此项目作为针对NMDA受体酒精作用位点新型特效药物研发而开展的前期基础研究，其结果为探寻NMDA 受体上酒精作用的药物靶点及相关研究提供了一定的理论依据。