REGULATION OF OSTEOCLAST VECTORIAL PROTON TRANSPORT

破骨细胞在很大程度上负责有序的新陈代谢 和骨骼的重塑，这是结构性的 完整性，并保持超过99%的身体储存的钙。 我的实验室提出了一种矢量传输的机制， 质子进入骨吸收室， 作为骨吸收调节靶点的大分子成分。 在我们纯化的这些皱褶膜氯离子通道中， 限制骨吸收质子运输的组分。 我们 正在提议研究荷尔蒙和遗传调节的 氯离子通道活性在破骨细胞的分化和 成熟的骨吸收细胞。 具体而言，我们建议： 1.使用以下方法表征62 kDa皱褶膜氯离子通道： 生物化学和电生理技术，以：a.得到氨基酸 来自纯化的通道蛋白的肽的序列，B.建立 氯化物的免疫定位和纯化方法 海峡角激素调节和原位磷酸化相关 有氯离子通道活性 2.分离编码62 kD皱褶膜的cDNA和基因组克隆 氯离子通道，并使用这些序列：a.表征表达式 在培养的破骨细胞前体细胞中， 对已知影响破骨细胞功能的激素的反应， 分化，B.确定氯化物调节的相关要点 channel expression，c.地图控制区域所需的诱导 在分化过程中的表达和对激素 刺激，和d.使用衍生自 氯离子通道基因选择性抑制氯离子的表达 通道中发展破骨细胞的文化，并确定影响 这种对骨吸收的抑制。

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.