Na/Bicarbonate Cotransporters in Brain

许多酸碱转运蛋白参与脑内非细胞和细胞外pH的调节。Na/BicarbonaticCotranspl 11 crs（NBes）是一种重要的神经元电生理活性物质，它能改变神经元的pH值。虽然多种NBC变体在脑和其他组织中的生理学意义尚不清楚，但这些变体中的许多变体表现出不同的调节谱。NBCs由涉及蛋白激酶A和C以及相互作用蛋白的经典第二信使调节。然而，涉及磷脂的其他调节途径的作用尚未阐明。'111 e长期 本提案的目的是确定和表征Na+耦合位的调节机制：carbonatt！本研究旨在研究多种13 T转运蛋白（BT）的功能，以了解多种13 T的意义和转运蛋白功能的最新研究进展。膜片钳技术和荧光成像将用于检查磷脂酰肌醇4，S-磷酸（PIP：！）pHi和ncid挤出机的调节（例如，NBC、Nadrivcn CI-HCO，j交换器（NDCD E）和）。α-H交换体（NHEs）在培养的大鼠星形胶质细胞和海马神经元中的作用（目的I）。通过直接灌注，将改变细胞中的胞内P1 P ~+水平。B~ 1与细胞转染剂PIP-孵育，并用磷酸肌醇酶转染。更改单元格PIP”将<lsst！通过质谱和蛋白质-脂质重叠测定来检测。待检查的特征 包括PIP_2 ~ EC 50、Na~+和电压依赖性，以及对电荷转移剂和PIP_2阻滞剂敏感性。n tibodies.根据初步数据，PIP通过调节NBCe 1变体的细胞质氨基末端的调节作用来刺激NBCe 1变体的活性（Aim 2）。在目的2中，将使用异源表达截短的mel突变体的爪蟾（Xenopus）的双电极电压-阻尼和宏补丁技术来鉴定和表征调节转运蛋白F和PIP敏感性的N末端区域/残基。将产生PIP ~-剂量反应曲线。PIP的效果：！基于生物物理性质（例如，KM瓦尔值表示离子的迁移率，VIII ~.~将评估NBCC 1变体的电流-电压关系）。将评估含有鉴定的调节区的肽对NBC融合的影响。这些目标的结果将阐明PIP 2作为一种新的NBC活性调节机制，可能为调节神经元兴奋性的PIP 2靶点提供允许的pH环境。表征P[P2]对NBCel的刺激的分子基础扩展了我们对tht！多个NBCs的重要性，并提供了机制和结构的见解，以~MC功能。信息\，; 1.增强我们对脑细胞酸碱处理的理解，特别是与缺血、缺氧/缺氧、斯特罗和再灌注损伤相关的酸碱紊乱。

Many acid-base transporters contribute to inll'acellular and extracellular pH regul ation in brain. Electroge nic Na/Bicarbonatc Cotransp0l1crs (NBes) arc particu larly imp0l1ant because they al ter pH in response to neuronal activity. While the physiologic significance of multiple NBC vnriants in brain and other tissues is not known, many of these variants exhibit different regulatory profiles. NBCs are regulated by classic second messengers involving protein kinases A and C, as well as interacting proteins. However, the role of other regulatory pathways involving phospholipids for instance has 110t been elucida ted. '111e longterm objective of this proposal is to identify and characterize the regulatory mechanisms of Na+-coupled bit:arbonatt! transporters (BTs) in an effort to understand the significance of multiple 13Ts nnd the Illo leculnl'u'lsis of tramipo rler function. Patch-clamp techniques and fluo rcsccnce imaging will he used to exam inc phosphatidylinositol 4,S- hisphosphnte (PIP:!) regulation of pHi and ncid extrudcrs (e.g., NBCs, the Nadrivcn CI-HCO,j exchanger (NDCD E), and )./a-H exchangers (NHEs)) in cult ured astroc)'tes and neurons from rat hippocampus (Aim I). Int racellular P I P ~ levels will be altered in cells by di rect perfusion. b~lth incubation with cell ~ p e r m cant PI P", and transfection with phosphoinositide enzymes. Changes in cell ular PIP" will be <lsst!ssed by mass spectroscopy and a protein~ l ipid overlay assay. Characteristics to be examined include PJ P~ ECso, Na~ and voltuge dependencies, and sensitivity to charge scrccners and PIP2 blocJ.;ing nn tibodies. According to preliminary data, PIP" stimulates the activity of NBCe l va riants by modulating the rcgulatOlY role of thei r cytoplasmic amino termini (Aim 2). In Aim 2 , two-eleetrode voltage-damp and macropatch techniques with Xenopus ooC)"tes heterologously expressing trunca ted "mel mutant i\BCt'l va riants will be uscd to identify and characterize N terminal regions/ residues th at modulate transporter fUllction and PIP:: sensiti\ity. PIP ~ dose-response eurves will be generated. The effect of PIP:! on the biophysical properties (e .g., KM val lies for tl'ansported ions, Vlll~.~ va lu~ s, and current-voltage relationships) of NBCCl variants will be assessed. The effect of peptides containing id enti fied rcglliatory regions on NBC fU ll ction will be 'lssessed. The results from these aims will elucidate PIP~ as a novel regulatory mechanism of NBC activi ty that likely provi des a permissi\'c pH environment for PIP2 targets that modulate neuronal t'xcitahility. Characterizing the molecular basis of P[P2's sti mulation of NBCel extends our lmderstanding of tht! importance of multiple NBCs, and provides mechanistic and st ructural insight in to ~mc function. The information \,;;1\ enhance our understa nding of acid -base handling by brain cells, partieul arly in acid-base disturbances associ:Jted \,;;th ischemia, anoxia/ hypoxia, stroh, and reperftlsion inj ury.