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：
8419239
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 2016-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8419239
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 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 public health relevance 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 A 型嵌入细胞 (A-IC) 主动分泌质子，是调节血液 pH 值的关键角色。然而，人们对 A-IC 检测系统 pH 变化的机制知之甚少。质子分泌是由 V-ATP 酶实现的，V-ATP 酶功能失调会导致远端肾小管酸中毒和肾结石等严重疾病。在寻找潜在的酸/碱传感器时，我们发现附睾中的 A-IC 和类似的质子分泌细胞（透明细胞；CC）通过 sAC（一种碳酸氢盐激活的腺苷酸环化酶）感知管腔碳酸氢盐。该提案现在研究 A-IC 如何感知尿液 pH 值以及碳酸氢盐来调节质子分泌。一些嘌呤能受体被提议作为 pH 传感器，目标 1 将测试 A-IC 通过这些受体感知尿液 pH 变化的假设。拟议的实验基于我们的新数据，该数据表明管腔 ATP 刺激 CC 分泌质子。我们将鉴定仅在 A-IC 中表达的 P1 和 P2 受体，并检查它们在调节 V-ATP 酶依赖性质子分泌中的作用。我们还将检查 A-IC 的 ATP 分泌以及 CFTR 在此过程中的作用。通过 FACS 从 B1-EGFP 小鼠、C11 嵌入细胞系和显微解剖 OMCD 分离的 A-IC 将使用互补成像、生化和电生理程序进行检查。目标 2 将探讨 V-ATP 酶本身可能是 A-IC 的 pH 传感特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 是否是一种 pH 传感器，通过其胞质 V1 与其跨膜 V0 结构域的关联来调节 V-ATP 酶。我们将使用附睾作为可以在体内调节腔内 pH 值的模型系统。 V-ATPase 组装/拆卸将使用共聚焦显微镜、EM、co-IP 和细胞分级进行检查。我们将通过使用 FACS 分离的 A-IC 和体外灌注的 OMCD 将我们的发现“转化”为 A-IC。这些实验将有助于我们更好地了解肾腔酸化，这对于将血液 pH 值维持在非常狭窄的可行范围内至关重要。