Na/Bicarbonate Cotransporters in Brain

描述(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。为了同时调节细胞内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碱(如重碳酸盐)输送到它们的膜上。碳酸氢盐偶联转运蛋白(NBCs)是大脑pH的特别有效的调节器，而电生成转运蛋白在促进活性诱发的pH变化方面占据了不同寻常的利基。随着其中许多蛋白质的分子鉴定，我们对这一蛋白质家族的了解已经扩大。这一建议的长期目标是阐明脑内多个产生电的NBCs的生理学意义。针对特定亚型的抗体将被用来检查这些蛋白质在整个大鼠大脑中的表达情况(目标1)。随后，将探讨每个NBC对大鼠海马区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活性的鉴定和生物物理特性(目标2)。因此，功能特性将被指定给目标1中确定的分子。最后，为了阐明目标2中所研究的NBCe1蛋白的结构特征，将研究NBCe1的结构-功能关系(目标3)。利用有关阴离子交换器(AE)的现有信息，将确定负责离子结合和转运的特定区域和残基，以及pH和电压敏感性。嵌合的NBC-AEs和截短/突变的NBCs将在卵母细胞中使用微电极和宏斑片技术进行表达和功能表征，以及在转基因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传输，以及更详细的离子传输特性、pH和电压依赖关系以及活化动力学。结果将提供与超级家族其他成员相关的信息。了解产生电的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，以及它们参与癫痫、缺血和缺氧等酸碱失衡的过程。

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.