ION CHANNELS AND SIGNALING MECHANISMS IN T LYMPHOCYTES

细胞内游离钙([Ca~(2+)]i)水平的升高 在受体驱动的多种细胞类型的激活中发挥中心作用， 包括T淋巴细胞。在T细胞中，由T细胞识别抗原 受体触发细胞内钙释放并刺激 Ca~(2+)通过质膜通道内流。新生理学 针对单细胞水平的方法已经揭示了复杂性 在这种反应中，如[Ca~(2+)]i振荡，并提供了直接 有证据表明不同类别的钙离子和钾离子通道对 钙离子信号转导过程。 这项提议的长期目标是理解分子 在T细胞激活过程中调节钙信号的机制。这个 提出的实验特别寻求定义这些特性， T细胞内钙离子通道的调节和分布及其意义 钾通道在钙信号转导中的作用。荧光视频- 成像和膜片钳记录方法将结合在一起来表征 受体调节的钙离子通道的药理作用 离子选择性和单通道特性。亚细胞 将抗原受体与钙通道激活联系起来的机制 在很大程度上是未知的。药理探针将被应用于区分 肌醇磷酸盐和细胞内钙库在细胞内调节中的作用 钙通道激活。视频显微镜的进步使它 有可能以高时间和空间成像细胞内钙离子 以单元格为单位的分辨率。我们将使用这些技术来探索 钙离子通道和抗原受体之间的物理联系，通过 封闭膜上抗原受体后钙离子内流部位的可视化 细胞表面。用最近发现的高度特异的K+-进行实验 通道阻滞剂揭示电压门控和钙激活的K+通道 参与钙离子信号的振荡，可能是通过影响 膜电位。我们寻求建立K+通道之间的联系， 膜电位和[Ca~(2+)]_i在电压和钙离子成像实验中的应用 依赖于钙离子的染料和笼状钙离子的光解。 通过它们对T细胞钙信号的影响，钙离子和钾离子通道 代表调节免疫反应的敏感控制点。 通过扩展我们对离子通道属性和调节的知识 这些机制的研究可能会导致识别和合理性 设计控制T细胞反应性的药物。通过这种方式， 拟议中的研究可能会发现未来治疗应用的范围很广 人类疾病，包括免疫缺陷，如艾滋病，自身免疫 疾病、过敏、移植排斥反应和与急性排斥相关的疾病 和慢性炎症。

An increase in the level of intracellular free calcium ([Ca2+]i) occupies a central role in the receptor-driven activation of many cell types, including T lymphocytes. In T cells, antigen recognition by the T cell receptor triggers Ca2+ release from intracellular stores and stimulates Ca2+ influx through channels in the plasma membrane. New physiological approaches directed at the level of single cells have revealed complexities in this response, such as [Ca2+]i oscillations, and have provided direct evidence for distinct classes of Ca2+ and K+ channels that contribute to the Ca2+ signaling process. The long-term objectives of this proposal are to understand the molecular mechanisms that regulate Ca2+ signaling during T-cell activation. The proposed experiments specifically seek to define the properties, regulation, and distribution of Ca2+ channels in T cells and to understand the role of K+ channels in modulating Ca2+ signaling. Fluorescence video- imaging and patch-clamp recording methods will be combined to characterize receptor-regulated Ca2+ channels in terms of their pharmacological profile, ionic selectivity, and single-channel properties. The subcellular mechanisms that link the antigen receptor to activation of Ca2+ channels are largely unknown. Pharmacological probes will be applied to distinguish the role of inositol phosphates and intracellular Ca2+ stores in mediating Ca2+-channel activation. Advances in video microscopy have made it possible to image intracellular Ca2+ with high temporal and spatial resolution in single cells. We will use these techniques to explore the physical association between Ca2+ channels and the antigen receptor, by visualizing sites of Ca2+ influx after capping the antigen receptor on the cell surface. Experiments with recently identified, highly specific K+- channel blockers reveal that voltage-gated and Ca2+-activated K+ channels contribute to oscillatory Ca2+ signaling, possibly through an influence on the membrane potential. We seek to establish the link between K+ channels, membrane potential, and [Ca2+]i in imaging experiments using voltage- and Ca2+-dependent dyes and photolysis of caged Ca2+. Through their influence on Ca2+ signaling in T cells, Ca2+ and K+ channels represent sensitive control points for modulation of the immune response. By extending our knowledge of ion channel properties and regulatory mechanisms, these studies may lead to the identification and rational design of drugs to control T-cell responsitivity. In this way, the proposed research may find future therapeutic application to a wide variety of human diseases, including immunodeficiencies such as AIDS, autoimmune disorders, allergy, graft rejection, and disorders associated with acute and chronic inflammation.