STRUCTURE AND REGULATION OF OSTEOBLAST CALCIUM CHANNELS

描述(改编自申请人的摘要)：长期目标 这项研究建议是为了了解表情是如何变化的，或者 成骨细胞中钙通道的调节影响骨代谢。 最近的研究表明，钙通道的激活在 成骨细胞是许多骨骼调节因子的共同特征 细胞内钙信号在骨调控中的重要作用 改建。然而，其分子结构和细胞调控 成骨细胞的钙通道在很大程度上仍未明确。之前的研究已经 证实了钙通道的三种异构体在 成骨样骨肉瘤细胞(α1S、α1C和α1D)，以及 甲状旁腺激素选择性地激活α1D亚型。这个 这项研究的目的是更全面地描述结构， 钙通道α1D异构体的功能与调控 成骨细胞。具体目标是：1)确定分子 α1D亚型的结构；2)检查时间和激素 成骨细胞分化过程中α1D基因表达的调控 明确甲状旁腺激素激活α1D的机制；4) 确定Alpha1D信号介导的特定功能效应 途径；5)检测其他激素对α1D活性的调节 还有毒品。钙通道基因克隆将被分离、测序和使用 用于mRNA水平的定量分析。钙通道活性将 被评估(使用荧光探头)以测量 细胞内钙，以及放射性同位素摄取试验来测量 钙离子内流。特异性钙通道拮抗剂以及反义 技术，将被用于检查功能角色和荷尔蒙 成骨细胞钙通道的调节。有人建议，这一结果 将提供新的和重要的信息 关于成骨细胞钙的分子结构和细胞调控 经络及其在骨代谢中的作用，以及提供洞察力 研究骨调节激素的细胞作用机制。这些 洞察力对于理解调节骨骼的事件至关重要 重塑，并最终为制定有效的治疗方案 治疗骨病的方法，包括骨质疏松症。

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.