Na/Bicarbonate Cotransporters in Brain

脑中的钠/碳酸氢盐协同转运蛋白

• 批准号: 6677218
• 负责人: MARK Oliver BEVENSEE
• 金额: $ 29.97万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-15 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6677218
• 关键词: Xenopus oocyte; acidity /alkalinity; bicarbonates; biological transport; brain; brain cell; chemical kinetics; fluorescence microscopy; hippocampus; immunochemistry; laboratory rat; membrane potentials; membrane transport proteins; microelectrodes; protein isoforms; protein localization; protein quantitation /detection; protein structure function; recombinant proteins; site directed mutagenesis; sodium; tissue /cell culture; transfection; voltage /patch clamp; western blottings

DESCRIPTION (provided by applicant): In brain, pH regulation is important because many ion channels, neurotransmitter-uptake systems, and cellular processes are sensitive to changes in pH. To regulate both intracellular pH (pHi) and extracellular Ph (pHo), brain cells such as neurons and glia utilize plasma-membrane transporters to shuttle hydrogen and bases such as bicarbonate across their membranes. Bicarbonate-coupled transporters such as Na/Bicarbonate Cotransporters (NBCs) are particularly potent regulators of brain pH, and electrogenic ones occupy an unusual niche in contributing to activity-evoked changes in pH. Our understanding of this family of proteins has expanded following the molecular identification of many of these proteins. The long-term objective of this proposal is to elucidate the physiological significance of multiple electrogenic NBCs in brain. Antibodies to specific isoforms will be used to examine the expression profiles of the proteins throughout the rat brain (Aim 1). Subsequently, the contribution of each NBC to the pH physiology of the rat hippocampus will be explored. The identification and biophysical characterization of NBC activity in each hippocampal cell type will be revealed and compared using fluorescence imaging of pHi and patch-clamp techniques (Aim 2). Functional properties will therefore be assigned to the molecules identified in Aim 1. Finally, to elucidate the structural features that underlie the functional properties of the NBCe1 proteins studied in Aim 2, structure-function relationships of NBCe1 will be examined (Aim 3). Utilizing available information on related anion exchangers (AEs), specific regions and residues responsible for ion binding and translocation, as well as pH and voltage sensitivities will be determined. Chimeric NBC-AEs and truncated/mutant NBCs will be expressed and functionally characterized in oocytes using microelectrodes and the macropatch technique, as well as in transfected mammalian cells using fluorescence imaging. Studies will involve examining fundamental bicarbonate transport, as well as the more detailed transport properties of ion, pH, and voltage dependencies and activation kinetics. Results will provide information pertinent to other members of the superfamily. Understanding the expression, function, and structure of electrogenic NBCs will help clarify the influence of these proteins on neuronal activity and synaptic transmission, as well as their involvement in acid-base disturbances such as epilepsy, ischemia, and hypoxia.

描述（由申请人提供）：在脑中，pH调节是重要的，因为许多离子通道、神经递质摄取系统和细胞过程对pH的变化敏感。为了调节细胞内pH（pHi）和细胞外pH（pHo），脑细胞（如神经元和神经胶质细胞）利用质膜转运蛋白将氢和碱（如碳酸氢盐）穿梭穿过其膜。碳酸氢盐偶联转运蛋白，如Na/Bicarbonate Cotransporters（NBCs）是特别有效的调节脑pH值，和生电的占据了一个不寻常的利基在促进活动诱发的pH值的变化。我们的理解这个家庭的蛋白质已经扩大了许多这些蛋白质的分子鉴定。本研究的长期目标是阐明脑内多个生电NBCs的生理意义。特异性同种型的抗体将用于检查整个大鼠脑中蛋白质的表达谱（目的1）。随后，将探索每种NBC对大鼠海马pH生理学的贡献。将使用pHi荧光成像和膜片钳技术揭示和比较每种海马细胞类型中NBC活性的鉴定和生物物理表征（目的2）。因此，功能特性将归属于目标1中确定的分子。最后，为了阐明目标2中研究的NBCe 1蛋白的功能特性的结构特征，将检查NBCe 1的结构-功能关系（目标3）。利用相关阴离子交换剂（AE）的现有信息，将确定负责离子结合和易位的特定区域和残基，以及pH和电压敏感性。嵌合NBC-AE和截短/突变NBC将在卵母细胞中使用微电极和macropatch技术表达和功能表征，以及在转染的哺乳动物细胞中使用荧光成像。研究将涉及检查基本的碳酸氢盐运输，以及更详细的离子，pH值和电压依赖性和激活动力学的运输特性。研究结果将提供与该超家族其他成员相关的信息。了解生电NBCs的表达、功能和结构将有助于阐明这些蛋白质对神经元活动和突触传递的影响，以及它们参与酸碱失衡，如癫痫、缺血和缺氧。