CAPACITATIVE CALCIUM ENTRY AND STORES IN KERATINOCYTES

描述(改编自《调查者摘要》)：细胞内钙 钙离子浓度是角质形成细胞增殖的重要调节因子 和差异化。因此，[Ca~(2+)]i的变化在 角质形成细胞的功能与组织分化、伤口愈合、 癌症的发展和细菌的入侵。首席调查员发现 近年来，细菌入侵过程中发生了核内[Ca~(2+)]_n的变化 角质形成细胞。这些[Ca~(2+)]_n的变化也依赖于Ca~(2+)内流。 此外，存储耗尽引起的电容电流的大小为 这取决于角质形成细胞的分化状态。自.以来 [Ca2+]n的变化是ALL细胞基因表达的重要调节因素，如 变化可能在宿主对细菌的反应中发挥重要作用 入侵。然而，在了解这一入侵过程之前， [Ca~(2+)]_n的调控机制必须阐明。此外，它是 解释电容电流因角质形成细胞不同而变化的必要性 分化状态。本提案开始努力了解 这些[钙]调节机制。因此，这项建议有以下几个方面 具体目标：具体目标1建议确定细胞内钙离子如何 商店会影响核内[钙离子]。特定目标2建议对钙离子进行表征 由钙离子激活的通透性通道储存耗竭，并确定是否 由此产生的钙离子内流改变了细胞核的[钙离子]。具体目标3将 证明核内钙信号系统在 钙激活的分化角质形成细胞与未分化角质形成细胞的比较 角质形成细胞。[Ca~(2+)]i将用广域反褶积方法测量 使用Fura-2的显微镜；全细胞和单个细胞的钙离子通道特性 通道、膜片钳技术。诱导角质形成细胞分化 通过暴露于培养物中的Ca~(2+)，并通过诱导总蛋白和 细胞角蛋白13.完成拟议的具体目标将建立 电容激活钙电流的特性及钙储备量 影响分化和未分化角质形成细胞中的[钙]n，以及 它们与[Ca~(2+)]_n变化的关系可以影响基因表达。

DESCRIPTION (Adapted from the Investigator's Abstract): Intracellular calcium concentration ([Ca2+]i) is an important regulator of keratinocyte proliferation and differentiation. Hence, changes in [Ca2+]i play an important role in keratinocyte functions involved with tissue differentiation, wound healing, cancer development and bacterial invasion. The Principal Investigator found recently that nuclear [Ca2+] ([Ca2+]n) changes occur during bacterial invasion of keratinocytes. These [Ca2+]n changes were also dependent on Ca2+ influx. Moreover, the magnitude of the store depletion induced capacitative current was found to depend on the differentiation status of the keratinocyte. Since [Ca2+]n changes are important regulators of gene expression in all cells, such changes are likely to play an important role in the host response to bacterial invasion. However, before this invasion process can be understood, the mechanisms by which [Ca2+]n is regulated must be elucidated. In addition, it is necessary to elucidate why the capacitative current varies with keratinocyte differentiation state. The present proposal initiates efforts to understand these [Ca2+] regulatory mechanisms. Hence, this proposal has the following Specific Aims: Specific Aim 1 proposes to determine how intracellular Ca2+ stores affect nuclear [Ca2+]. Specific Aim 2 proposes to characterize the Ca2+ permeable channels activated by Ca2+ store depletion, and to determine whether the resulting Ca2+ influx alters nuclear [Ca2+]. Specific Aim 3 will demonstrate that the nuclear Ca2+ signaling system is altered in Ca2+-activated, differentiated keratinocytes as compared to no differentiated keratinocytes. [Ca2+]i will be measured with wide-field, deconvolution microscopy using fura-2; Ca2+ channel properties with whole-cell and single channel, patch clamp techniques. Keratinocyte differentiation will be induced by exposure to Ca2+ in culture, and monitored by induction of involucrin and cytokeratin 13. Completion of the proposed Specific Aims will establish the characteristics of the capacitatively activated Ca current and the Ca stores that affect [Ca2+]n in differentiated and undifferentiated keratinocytes, and their relationships to the [Ca2+]n changes that can affect gene expression.