Structure and Function in NSS transporters

NSS 转运蛋白的结构和功能

• 批准号: 7458719
• 负责人: GARY W RUDNICK
• 金额: $ 29.46万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7458719
• 关键词: Address; Amino Acid Transporter; Bioenergetics; Condition; Coupled; Coupling; Crystallography; Dopamine; Escherichia coli; Family; Genetic Screening; Glycine; Homo; Indium; Ions; Learning; Mediating; Methods; Mutation; Nature; Neurotransmitters; Norepinephrine; Organism; Preparation; Process; Proteins; Recycling; Role; Serotonin; Site; Sodium; Structural Protein; Structure; Testing; Transmembrane Domain; Tryptophan; crosslink; design; extracellular; gamma-Aminobutyric Acid; genetic selection; member; neurotransmission; neurotransmitter release; neurotransmitter uptake; novel strategies; protein structure; proteoliposomes; reconstitution; research study; stoichiometry; symporter; tool; two-dimensional; uptake

DESCRIPTION (provided by applicant): The neurotransmitter sodium symporter (NSS) family includes transporters responsible for the re-uptake of neurotransmitters GABA, glycine, norepinephrine, serotonin, and dopamine. They represent a key element in neurotransmission whereby the extracellular concentration of neurotransmitter is regulated at a low level, and transmitters are efficiently recycled. Also in this family are many transporters for amino acids not involved in neurotransmission, including many predicted prokaryotic proteins whose function has not yet been identified. A major limitation in understanding these proteins has been the difficulty in expressing and purifying significant amounts of protein for structural and functional studies. The recent demonstration that 1 of the prokaryotic NSS sequences encodes a functional tryptophan transporter (TnaT) has opened up new avenues for understanding the structure and function of all of these transporters. This proposal is designed to take advantage of these new approaches to understand TnaT and to relate these findings to mammalian neurotransmitter transporters. The proposed experiments include an examination of the bioenergetics of sodium-coupled tryptophan transport. These experiments will determine which ion gradients are coupled to tryptophan transport and will establish the stoichiometry of transport. The proposal also describes the use of genetic selection methods to estimate the physical proximity between different regions of the protein structure. Finally, there are experiments to evaluate the possibility that TnaT functions as a homo-oligomer and to gain structural information about the transporter using two-dimensional (2D) crystallography.

描述（由申请人提供）：神经递质钠同向转运蛋白（NSS）家族包括负责重新摄取神经递质 GABA、甘氨酸、去甲肾上腺素、血清素和多巴胺的转运蛋白。它们代表了神经传递中的关键要素，神经递质的细胞外浓度被调节在较低水平，并且递质被有效地回收。该家族中还有许多不参与神经传递的氨基酸转运蛋白，包括许多功能尚未确定的预测原核蛋白。理解这些蛋白质的一个主要限制是难以表达和纯化大量蛋白质以进行结构和功能研究。最近证明原核 NSS 序列之一编码功能性色氨酸转运蛋白 (TnaT)，为理解所有这些转运蛋白的结构和功能开辟了新途径。该提案旨在利用这些新方法来理解 TnaT 并将这些发现与哺乳动物神经递质转运蛋白联系起来。拟议的实验包括对钠偶联色氨酸转运的生物能学的检查。这些实验将确定哪些离子梯度与色氨酸运输耦合，并将建立运输的化学计量。该提案还描述了使用遗传选择方法来估计蛋白质结构不同区域之间的物理接近度。最后，还有实验评估 TnaT 作为同源低聚物的可能性，并使用二维 (2D) 晶体学获得有关转运蛋白的结构信息。