Na/Bicarbonate Cotransporters in Brain

脑中的钠/碳酸氢盐协同转运蛋白

• 批准号: 7052894
• 负责人: MARK Oliver BEVENSEE
• 金额: $ 26.9万
• 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/7052894
• 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）：在大脑中，pH 调节很重要，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)，神经元和神经胶质细胞等脑细胞利用质膜转运蛋白将氢和碳酸氢盐等碱穿过其膜。碳酸氢盐偶联转运蛋白，如 Na/碳酸氢盐协同转运蛋白 (NBC)，是大脑 pH 值特别有效的调节剂，而产电转运蛋白在促进活动引起的 pH 值变化方面占据着不寻常的地位。随着许多蛋白质的分子鉴定，我们对这个蛋白质家族的了解得到了扩展。该提案的长期目标是阐明大脑中多种生电 NBC 的生理意义。特定亚型的抗体将用于检查整个大鼠大脑中蛋白质的表达谱（目标 1）。随后，将探讨每种 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来揭示和比较每种海马细胞类型中 NBC 活性的识别和生物物理特征（目标 2）。因此，功能特性将分配给目标 1 中鉴定的分子。最后，为了阐明目标 2 中研究的 NBCe1 蛋白功能特性背后的结构特征，将检查 NBCe1 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 将使用微电极和大贴片技术在卵母细胞中表达并进行功能表征，以及使用荧光成像在转染的哺乳动物细胞中进行表达和功能表征。研究将涉及检查基本的碳酸氢盐传输，以及离子、pH 值、电压依赖性和活化动力学的更详细的传输特性。结果将提供与该超家族其他成员相关的信息。了解生电 NBC 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，以及它们与癫痫、缺血和缺氧等酸碱紊乱的关系。