ION CHANNELS AND SIGNALING MECHANISMS IN T LYMPHOCYTES

细胞内游离钙（[Ca 2 +]i）水平的增加占据了细胞内钙离子浓度的一个重要部分。 在许多细胞类型的受体驱动激活中起中心作用， 包括T淋巴细胞。 在T细胞中， 受体触发Ca 2+从细胞内储存释放，并刺激 Ca 2+通过质膜中的通道内流。 新的生理 针对单细胞水平的方法揭示了复杂性， 在这种反应中，如[Ca 2 +]i振荡，并提供了直接的 不同类别的Ca 2+和K+通道的证据有助于 Ca 2+信号传导过程。 这项提案的长期目标是了解分子 在T细胞活化过程中调节Ca 2+信号传导的机制。 的 所提出的实验特别地试图定义这些性质， 调节和T细胞中Ca 2+通道的分布，并了解 K+通道在调节Ca 2+信号中的作用。 荧光视频- 将结合成像和膜片钳记录方法来表征 受体调节的Ca 2+通道的药理学特征， 离子选择性和单通道特性。 亚细胞 将抗原受体与Ca 2+通道激活联系起来的机制 大部分都是未知的。 将应用药理学探针来区分 肌醇磷酸和细胞内钙库在介导 Ca 2+通道激活。 视频显微镜技术的进步 可能以高的时间和空间分辨率对细胞内Ca 2+进行成像 单细胞的分辨率。 我们将使用这些技术来探索 Ca 2+通道和抗原受体之间的物理关联， 在给抗原受体加帽后，使Ca 2+内流的位点可视化， 细胞表面 最近发现的高特异性K+- 通道阻断剂显示电压门控和Ca 2+激活的K+通道 有助于振荡Ca 2+信号，可能是通过影响 膜电位。 我们试图建立K+通道之间的联系， 膜电位和[Ca 2 +]i在成像实验中使用电压和 钙离子依赖性染料和笼状钙离子的光解。 通过影响T细胞中的Ca 2+信号，Ca 2+和K+通道 代表调节免疫应答的敏感控制点。 通过扩展我们对离子通道特性和调控的知识， 机制，这些研究可能会导致识别和合理的 控制T细胞反应性的药物设计。 如此则 拟议的研究可能会发现未来的治疗应用，以广泛的各种 人类疾病，包括免疫缺陷，如艾滋病，自身免疫 疾病、变态反应、移植排斥和与急性 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.