Na/Bicarbonate Cotransporters in Brain

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的变化都很敏感。为了调节细胞内pH （pHi）和细胞外pH (pHo)，脑细胞（如神经元和胶质细胞）利用质膜转运蛋白将氢和碱（如碳酸氢盐）穿过它们的膜。碳酸氢盐偶联转运蛋白，如钠/碳酸氢盐共转运蛋白（NBCs）是脑pH的特别有效的调节因子，而电致蛋白在促进活性诱发的pH变化方面占据了不寻常的位置。随着许多这些蛋白的分子鉴定，我们对这一蛋白家族的理解已经扩大。本研究的长期目标是阐明脑内多发电致nbc的生理意义。针对特定同种异构体的抗体将用于检查整个大鼠大脑中蛋白质的表达谱（目的1）。随后，将探讨每种NBC对大鼠海马pH生理的贡献。利用pHi的荧光成像和膜片钳技术，将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特征（目的2）。因此，功能特性将分配给Aim 1中鉴定的分子。最后，为了阐明在Aim 2中研究的NBCe1蛋白功能特性的结构特征，将研究NBCe1的结构-功能关系（Aim 3）。利用有关阴离子交换剂（AEs）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和截断/突变nbc将在卵母细胞中使用微电极和巨贴片技术表达和功能表征，并在转染的哺乳动物细胞中使用荧光成像。研究将包括检查基本的碳酸氢盐运输，以及离子，pH值，电压依赖性和激活动力学的更详细的运输特性。结果将为超家族的其他成员提供相关信息。了解电致NBCs的表达、功能和结构将有助于阐明这些蛋白对神经元活动和突触传递的影响，以及它们在酸碱紊乱（如癫痫、缺血和缺氧）中的作用。

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.