STRUCTURE AND REGULATION OF OSTEOBLAST CALCIUM CHANNELS

描述（改编自申请人的摘要）： 这项研究的目的是了解表达的变化，或 成骨细胞中钙通道的调节影响骨代谢。 最近的研究表明，钙通道的激活， 成骨细胞是许多骨调节因子的共同特征，这表明 细胞内钙信号在骨调控中的重要作用 重塑 然而，其分子结构和细胞调节 成骨细胞的钙通道在很大程度上仍不明确。 先前的研究 证明了钙通道的三种亚型表达于 成骨细胞样骨肉瘤细胞（α 1 S、α 1C和α 1D）， 甲状旁腺激素选择性激活α 1D同种型。 的 本研究的目的是更全面地表征结构， 钙通道α 1D亚型在 成骨细胞 具体目标是：1）确定分子 alpha 1D亚型的结构; 2）检查时间和激素 成骨细胞分化过程中alpha 1D基因表达的调控; 3） 确定甲状旁腺激素激活alpha 1D的机制; 4） 确定由alpha 1D信号传导介导的特定功能效应 途径;和5）检查其他激素对alpha 1D活性的调节 还有毒品 钙通道cDNA克隆将被分离、测序和使用 用于mRNA水平的定量分析。 钙通道活性将 评估（使用荧光探针）以测量 细胞内钙，以及放射性同位素摄取测定，以测量 钙内流 特异性钙通道拮抗剂，以及反义 技术，将被用来检查功能的作用和激素 成骨细胞钙通道的调节。 建议结果 这项研究将提供新的和重要的信息 关于成骨细胞钙离子的分子结构和细胞调节 通道及其在骨代谢中的作用，以及提供见解 骨调节激素的细胞作用机制。 这些 这些见解对于理解调节骨骼的事件至关重要， 重塑，并最终，为发展有效的治疗 治疗包括骨质疏松症在内的骨疾病的方法。

DESCRIPTION (Adapted from the Applicant's Abstract): The long-term goal of this research proposal is to understand how changes in the expression, or regulation of calcium channels in osteoblasts affect bone metabolism. Recent research indicating that the activation of calcium channels in osteoblasts is a common feature of many bone regulatory factors suggests a significant role for intracellular calcium signalling in the control of bone remodeling. However, the molecular structure and cellular regulation of osteoblast calcium channels remain largely undefined. Previous studies have demonstrated that three isoforms of calcium channels are expressed in osteoblast-like osteosarcoma cells (alpha1S, alpha1C and alpha1D), and that parathyroid hormone selectively activates the alpha1D isoform. The objective of this study is to more fully characterize the structure, function and regulation of the calcium channel alpha1D isoform in osteoblasts. The Specific Aims are to: 1) determine the molecular structure of the alpha1D isoform; 2) examine the temporal and hormonal regulation of alpha1D gene expression during osteoblast differentiation; 3) define the mechanism of alpha1D activation by parathyroid hormone; 4) determine the specific functional effects mediated by the alpha1D signaling pathway; and 5) examine the regulation of alpha1D activity by other hormones and drugs. Calcium channel cDNA clones will be isolated, sequenced and used for the quantitative analysis of mRNA levels. Calcium channel activity will be assessed (using a fluorescent probe) to measure the level of intracellular calcium, as well as a radioisotope uptake assay to measure calcium influx. Specific calcium channel antagonists, as well as antisense techniques, will be used to examine the functional roles and hormonal regulation of osteoblast calcium channels. It is suggested that the results of the proposed research study will provide novel and important information about the molecular structure and cellular regulation of osteoblast calcium channels and their role in bone metabolism, as well as providing insight into the cellular mechanisms of action of bone regulatory hormones. These insights are critical to an understanding of the events that regulate bone remodeling and, ultimately, for the development of effective therapeutic approaches for the treatment of bone diseases, including osteoporosis.