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

V-ATP 酶的 pH 感应和酸分泌激活

• 批准号: 8593292
• 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
• 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/8593292
• 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

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)：A型肾间质细胞(A-IC)活跃地分泌质子，是调节血液pH的关键角色。然而，A-IC检测系统性pH变化的机制尚不清楚。质子的分泌是通过V-ATPase实现的，而严重的疾病，如远端肾小管酸中毒和肾结石，则是由于V-ATPase功能障碍造成的。在寻找潜在的酸/碱感受器时，我们发现A-IC和附睾中类似的质子分泌细胞(Clear Cells；CCS)通过SAC感知鲁米那碳酸氢盐，SAC是一种由碳酸氢盐激活的腺苷环化酶。这项提议现在研究了A-IC如何感知尿液pH以及小苏打如何调节质子分泌。一些嘌呤能受体被认为是pH传感器，Aim 1将验证A-IC通过这些受体感知尿液pH变化的假设。拟议的实验是基于我们的新数据，该数据表明鲁米那ATP刺激CCS的质子分泌。我们将识别仅在A-IC中表达的P1和P2受体，并研究它们在调节V-ATPase依赖的质子分泌中的作用。我们还将研究A-ICs的ATP分泌和CFTR在这一过程中的作用。FACS从B1-EGFP小鼠、C11嵌入细胞系和显微解剖的OMCDs中分离的A-ICs将使用互补的成像、生化和电生理程序进行检测。目的2将探索V-ATPase本身可能是A-ICs pH敏感特性的一部分。这些实验基于令人兴奋的数据，表明V-ATPase A2亚单位是参与细胞内靶向事件的pH传感器。我们将研究“质膜”A4是否是一个pH传感器，它通过胞质V1和跨膜V0结构域的结合来调节V-ATPase。我们将使用附睾作为模型系统，在其中管腔pH可以在体内调节。V-ATPase的组装/拆解将使用共聚焦显微镜、EM、共IP和细胞分级进行检测。我们将通过使用FACS分离的A-IC和体外灌流的OMCD将我们的发现“转化”到A-IC上。这些实验将有助于我们更好地了解肾管腔酸化，这是将血液pH值维持在非常窄的可行范围内的核心。