REGULATION OF OSTEOCLAST VECTORIAL PROTON TRANSPORT

破骨细胞矢量质子运输的调节

• 批准号: 2081568
• 负责人: PAUL H SCHLESINGER
• 金额: $ 16.94万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2081568
• 关键词: antisense nucleic acid; cell differentiation; chickens; chloride channels; gene expression; genetic mapping; genetic regulation; hormone regulation /control mechanism; immunocytochemistry; immunoprecipitation; ion transport; molecular cloning; nucleic acid sequence; osteoclasts; phosphorylation; physiologic bone resorption; protein purification; tissue /cell culture

The osteoclast is in large part responsible for the orderly metabolism and remodeling of the bony skeleton which is required for structural integrity and maintains greater than 99% of the bodies stores of calcium. My laboratory has proposed a mechanism for the vectorial transport of protons into the bone resorption compartment which defines specific macromolecular components as targets for regulation of bone resorption.. Of these ruffled membrane chloride channel which we have purified is a limiting component in the transport of protons for bone resorption. We are proposing to study both the hormonal and genetic regulation of the chloride channel activity in the differentiation of the osteoclast and in the mature bone resorbing cell. Specifically we propose to: 1. Characterize the 62 kDa ruffled membrane chloride channel using biochemical and electrophysiological techniques to: a. obtain amino acid sequence of peptides from the purified channel protein, b. establish method for the immunolocalization and purification of the chloride channel, c. correlate hormonal regulation and in situ phosphorylation with chloride channel activity. 2. Isolate cDNA and genomic clones encoding the 62 kD ruffled membrane chloride channel and use these sequences to: a. characterize expression at RNA and protein levels in cultured osteoclast precursors and in response to hormones known to effect osteoclast function and differentiation, b. identify relevant points of regulation of chloride channel expression, c. Map controlling regions necessary for induction of expression during differentiation and in response to hormonal stimulation, and d. use antisense oligonucleotides derived from the chloride channel gene to selectively suppress expression of the chloride channel in developing osteoclasts in culture and determine the effect of such suppression on bone resorption.

破骨细胞在很大程度上负责有序的新陈代谢 和骨骨架的重塑，这是结构所需的 保持正直，并保持99%以上体内储存的钙。 我的实验室已经提出了一种机制，用于 质子进入骨吸收隔室，它定义了特定的 作为骨吸收调节靶点的大分子成分。 在我们纯化的这些褶皱的膜氯离子通道中， 骨吸收质子运输中的限制成分。我们 正在提议研究荷尔蒙和基因调节 氯离子通道活性在破骨细胞分化中的作用 成熟的骨吸收细胞。 具体来说，我们建议： 1.62 kDa皱褶膜氯离子通道的特性 生化和电生理技术：A.获得氨基酸 纯化的通道蛋白B.estably的多肽序列 氯化物的免疫定位和纯化方法 通道，C.相关激素调节和原位磷酸化 具有氯离子通道活性。 2.分离编码62kD褶皱膜的cDNAs和基因组克隆 氯通道，并使用这些序列来：A.表征表达 在RNA和蛋白质水平上培养的破骨细胞前体细胞和 对已知影响破骨细胞功能的激素的反应 辨析，B.找出氯离子的相关调节点 通道表达，c.诱导MAP所需的控制区 分化过程中的表达及其对激素的反应 刺激，以及D.使用源自 氯离子通道基因选择性抑制氯离子的表达 在体外培养破骨细胞中的作用及影响因素 这种对骨吸收的抑制。