Na/Bicarbonate Cotransporters in Brain

许多酸碱转运体参与脑内、外pH的调节。电化学钠/重碳酸盐共转移物(NBEs)对神经元活动有明显的抑制作用，因为它们改变了pH。虽然脑和其他组织中多个NBC变异体的生理意义尚不清楚，但这些变异体中的许多都表现出不同的调控特征。NBCs受经典的第二信使调控，涉及蛋白激酶A和C以及相互作用的蛋白质。然而，其他涉及磷脂的调节通路的作用还没有被阐明。‘111E长期 本研究的目的是鉴定和表征钠离子偶联的BIT的调控机制：Arbonatt！转运蛋白(BTS)，以了解多个13T的意义和短暂性呼吸功能的变化。膜片钳技术和荧光成像技术将用于检测磷脂酰肌醇4，S-组氨酸(PIP：！)调节培养的星形细胞和大鼠海马神经元(如NBCs、Nadrivcn CI-HCO、j交换器(NDCD E)和/a-H交换器(NHEs))的phi和nCID排泄物(如NbCs、Nadrivcn CI-HCO、J交换器(NDCD E)和/a-H交换器(NHEs))(Aim I)。直接灌流将改变细胞内的PI P~水平。与细胞孵育，并用磷脂酶转染法。细胞内P IP的变化将通过质谱学和蛋白质~L IPID重叠分析来检测。 包括PJ、P~ECSO、Na~和电压依赖性，以及对电荷调节剂和PIP2阻滞剂的敏感性。根据初步数据，PIP“通过调节胞质氨基末端的调节作用来刺激NbCe L变异体的活性(目标2)。在目标2中，利用双电极电压阻尼法和大斑片技术结合非洲爪哇(Xenopus OOC)“TES”异源表达截短的MEL突变体I\bct‘l变异体来鉴定和表征N个末端区域/残基对转运蛋白功能和PIP：：感觉的调节作用。将生成PIP~剂量-反应曲线。PIP的效果：！将评估NbCCl变体的生物物理性质(例如，转运离子的Km值，Vll~.~va Lu~S，以及电流-电压关系)。本文综述了含有免疫活性区域的多肽对NBC功能的影响。这些目标的结果将阐明PIP~作为一种新的NBC活性调节机制，可能为调节神经元毒性的PIP2靶点提供一个允许的pH环境。对P[P2‘S模拟NBCel的分子基础的表征，扩大了我们对它的理解！多个NBC的重要性，并提供了对TO~MC功能的机械和结构方面的见解。这些信息加强了我们对脑细胞酸碱处理的了解，特别是在酸碱失衡方面：缺血、缺氧/缺氧、缺氧、缺氧和再灌流。

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.