pH-sensing and activation of acid secretion by the V-ATPase

V-ATP 酶的 pH 感应和酸分泌激活

基本信息

批准号：
8777956
负责人：
SYLVIE BRETON
金额：
$ 37.85万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-12-15 至 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8777956
关键词：
ATP Receptors Acid-Base Equilibrium Acidosis Acids Adenosine Adenylate Cyclase Alanine Apical Architecture Bicarbonates Biochemical Biological Biological Assay Biological Models Blood Calcium Cell Culture Techniques Cell Fractionation Cell Line Cell membrane Cells Clear Cell Co-Immunoprecipitations Confocal Microscopy Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Diethyl Pyrocarbonate Disease Distal renal tubular acidosis Type 1 Duct (organ) structure Epididymis Evaluation Event Feedback Goals Health Histidine Homeostasis Image Immunofluorescence Immunologic In Situ Hybridization In Vitro Individual Intercalated Cell Kidney Kidney Calculi Knowledge Label Life Luciferases Maintenance Measures Mus Mutate Mutation Pathology Pathway interactions Peptides Procedures Process Property Protein Isoforms Protons Purinoceptor Rattus Regulation Renal function Research Personnel Reverse Transcriptase Polymerase Chain Reaction Role Signal Transduction Testing Tissues Transfection Translating Translations United States National Institutes of Health Urine Variant Western Blotting apical membrane base cellular microvillus equilibration disorder extracellular in vivo interdisciplinary approach intravital microscopy kidney cell luciferin mutant programs receptor research study response sensor time use urinary vacuolar H+-ATPase

项目摘要

DESCRIPTION (provided by applicant): Renal type A intercalated cells (A-ICs) actively secrete protons and are key players in the regulation of blood pH. However, the mechanisms by which A-ICs detect sysmetic pH variations are poorly understood. Proton secretion is achieved by the V-ATPase, and severe diseases such as distal renal tubular acidosis and kidney stones result from dysfunctional V-ATPase. While searching for potential acid/base sensors, we found that A-ICs and similar proton-secreting cells in the epididymis (clear cells; CCs) sense luminal bicarbonate via sAC, a bicarbonate-activated adenylyl cyclase. This proposal now examines how A-ICs sense urinary pH in addition to bicarbonate to regulate proton secretion. Some purinergic receptors were proposed as pH sensors, and Aim 1 will test the hypothesis that A-ICs sense urinary pH variations via these receptors. The proposed experiments are based on our new data showing that luminal ATP stimulates proton secretion by CCs. We will identify P1 and P2 receptors that are exclusively expressed in A-ICs, and we will examine their role in regulating V-ATPase-dependent proton secretion. We will also examine ATP secretion by A-ICs and the role of CFTR in this process. A-ICs isolated by FACS from B1-EGFP mice, the C11 intercalated cell line, and microdissected OMCDs will be examined using complementary imaging, biochemical and electrophysiological procedures. Aim 2 will explore the possibility that the V- ATPase itself might be part of the pH-sensing property of A-ICs. These experiments are based on exciting data showing that the V-ATPase a2 subunit is a pH sensor involved in intracellular targeting events. We will examine whether the "plasma membrane" a4 is a pH sensor that regulates V-ATPase via association of its cytosolic V1 with its transmembrane V0 domains. We will use the epididymis as a model system in which luminal pH can be modulated in vivo. V-ATPase assembly/disassembly will be examined using confocal microscopy, EM, co-IP and cell fractionation. We will "translate" our findings to A-ICs by using FACS isolated A-ICs and OMCDs perfused in vitro. These experiments will contribute to our better understanding of renal luminal acidification, which is central to the maintenance of blood pH within a very narrow viable range.

描述（由申请人提供）：肾脏型插入细胞（A-IS）积极分泌质子，并且是血液调节的关键参与者。但是，A-ICS检测系统pH变化的机制知之甚少。质子分泌是通过V-ATPase来实现的，严重的疾病，例如肾小管酸中毒和肾结石，由功能失调的V-ATPase引起。在搜索潜在的酸/碱传感器时，我们发现附睾（透明细胞； CCS）中的A-ICS和类似的质子分泌细胞通过SAC（一种碳酸氢盐激活的腺苷酸脂烯酰基环化酶）感知灯杆碳酸氢盐。现在，该提案研究了A-ICS除了碳酸氢盐外如何感知尿液pH来调节质子分泌。提出了一些嘌呤能受体作为pH传感器，AIM 1将检验以下假设，即A-IS通过这些受体介绍了尿液pH的变化。提出的实验基于我们的新数据，表明Luminal ATP刺激CCS的质子分泌。我们将确定仅在A-ICS中表达的P1和P2受体，并将研究它们在调节V-ATPase依赖性质子分泌方面的作用。我们还将检查A-ICS的ATP分泌以及CFTR在此过程中的作用。由B1-EGFP小鼠，C11插入细胞系和微分机OMCD分离的A-IS将使用互补成像，生化和电生理程序检查。 AIM 2将探讨V- ATPase本身可能是A-ICS pH值属性的一部分的可能性。这些实验基于令人兴奋的数据，表明V-ATPase A2亚基是参与细胞内靶向事件的pH传感器。我们将检查“质膜” A4是否是通过其胞质V1与跨膜V0结构域的关联来调节V-ATPase的pH传感器。我们将使用附睾作为模型系统，其中可以在体内调节腔pH。 V-ATPase组装/拆卸将使用共聚焦显微镜，EM，Co-IP和细胞分馏。我们将通过使用FACS孤立的A-ICS和OMCD在体外灌注的FACS将我们的发现“转化”到A-IS。这些实验将有助于我们更好地理解肾脏腔酸化，这对于在非常狭窄的可行范围内维持血液pH值至关重要。