中枢神经系统中谷氨酰胺跨膜转运的分子基础和调控

The sodium-coupled neutral amino acid transporters (SNATs) play many important physiological roles, for example in the glutamate-glutamine cycle in the brain and in cellular nitrogen metabolism. However, little is known about the actual mechanism and structural basis of glutamine transport by SNATs. One of the five known SNAT subtypes, SNAT2, will be studied because of its widespread distribution in almost all mammalian cells. The long-term goal of this proposal is to understand the mechanistic and structural bases of amino acid transport by SNAT2. In order to investigate this transport process, wild-type and mutant transporters will be expressed in mammalian cells，the topological stucture, cysteines functions in SNAT2, amino acid transport and regulaiton mechanism by extralcellular Cl- to SNAT2 transport will be studied by immunocytochemistry, patch clamping and substrate uptake, etc. Furthermore, The following hypotheses will be tested: 1) SNAT2 contains 11 transmembrane domains (TMD), the C terminus is extracellular and the N terminus is intracelluar. 2) Conserved cysteines in SNAT2 are important to SNAT2 structure and function. 3) TMD 1, 3, 6 and 8 are important to amino acid transport of SNAT2. 4) Amino acid and Na+ transport by SNAT2 are regulated by extracelluar anions. The research proposal will focus on four specific aims: 1) To determine topological structure of SNAT2, including locations of SNAT2 C-terminus and N terminus, the numbers of SNAT2 TMD. 2) To determine if 7 cysteines are important for SNAT2 sturcture and function, and if there is/are disulfide bond(s) in SNAT2. 3) To indentify structural elements of SNAT2 that are required for amino acid transport. 4) To determine whether extracellualr anions modulate amino acid transport by SNAT2. To identify structural elements of SNAT2 and specific amino acids in the TMDs of SNAT2 that are involved in controlling the functional properties of SNAT2 may contribute significantly to our understanding of the structure-function relationship and the transport mechanism of SNAT2 and other family's transporters.

中枢神经系统中，Na+偶联的中性氨基酸转运蛋白（SNATs）主要跨膜转运谷氨酰胺等小的中性氨基酸，在大脑和细胞氮代谢中具有重要的生理功能，与癫痫等疾病的发生密切相关。目前人们对SNATs跨膜转运谷氨酰胺的分子机制和结构基础所知甚少。本申请以SNATs家族中最为广泛分布的第二个亚型SNAT2为研究对象，将野生型及突变转运蛋白在哺乳动物细胞中表达，运用免疫细胞化学、膜片钳和底物氨基酸摄取等方法，系统地研究SNAT2的拓扑学结构、SNAT2分子中半胱氨酸的功能、影响SNAT2跨膜转运氨基酸的重要结构区域及细胞外阴离子对SNAT2转运氨基酸的调控等，揭示SNAT2的拓扑结构、氨基酸转运的分子机制、动力学模型及调控机制，为理解和研究其他转运蛋白结构和功能及其调控机制提供理论依据，为发现新的药物治疗相关神经系统疾病提供可靠依据。

利用免疫荧光、化学修饰、全细胞记录技术等细胞生物学、分子生物学和电生理学等研究方法，对SNAT2的分子结构基础和调控进行了系统研究。研究结果表明，SNAT2的拓扑结构与预测的不同，其N端和C端均位于细胞膜外，有10个跨膜区域（TMD），含有三个糖基化位点，位于TMD4和TMD5之间。SNAT2分子中含有一对二硫键，在C245和C279之间形成，此二硫键对SNAT2在细胞膜上的定位没有影响，对SNAT2转运氨基酸活性有一定影响，但不显著。SNAT2分子7个Cys中，C228和C303对SNAT2转运活性十分重要，C303参与了SNAT2与转运底物的结合。通过测定TMD1、3、6、8中保守氨基酸突变转运蛋白对Ala和Na+表观亲和常数，发现TMD1、6和8是SNAT2分子中重要的功能区域。测定了21个突变转运蛋白对阴离子渗漏电流的影响，发现SNAT2分子中H304、Y337和C-ter均能增加SNAT2对阴离子电流的渗漏作用，表明其可能参与了SNAT2对阴离子渗漏电流的调节，而N79、N82、G86、V302、A306、L308和T384严重降低了阴离子渗漏电流，表明这些氨基酸残基可能参与了抑制阴离子渗漏作用。以上研究丰富了人们对中枢神经系统中谷氨酰胺转运的过程和机制的了解，为发现治疗相关神经性疾病的靶向药物提供理论依据。