ROLE OF ION CHANNELS IN MONONUCLEAR PHAGOCYTE ACTIVATION

蛋白质磷酸化的相互作用，钙的增加和变化 在膜上，电位驱动单核巨噬细胞的激活。离子 巨噬细胞中的通道激活似乎调整了 激发细胞反应的激活信号，包括 分泌增强，颗粒吞噬和细胞毒性增强。离子 通道可以通过提供钙内流途径来触发响应， 他们可能会通过改变主宰 钙的内流，它们本身可能被调节和/或激活 通过钙和磷酸化。该提案的目标是研究 同时发出多个信号，目的是确定协同作用 增强吞噬细胞功能反应的相互作用。我们会 鉴定和表征电压不敏感的钙离子内流途径 由于激动剂结合而打开的吞噬细胞 到表面受体。我们将使用Fura-2荧光比率光度法 与全细胞和穿孔贴片相结合的视频成像 在单个细胞中将CaI与膜电流相关联的记录。 将进行实验，以确定通过以下方式直接激活 G蛋白、磷酸化或钙参与控制 受体操作的钙离子通透通道(ROCC)。一直以来 提示钙通过ROCCs的内流是由电学控制的 由K+和Cl-电导建立的驱动力，因此 我们将调查这些导体是否紧密耦合到 激动剂刺激后CAI的变化。我们将确定是否 神经肽和氯离子对细胞膜K+和Cl-电导的调节 激活的巨噬细胞释放的细胞因子或脂氧合酶代谢产物 调节蔡氏瞬变对表面刺激的反应。我们将使用 用于跟踪呼吸爆发活动的光子计数技术 单个吞噬细胞在颗粒刺激和可溶性刺激下均被激活。 我们将建立超氧化物释放、钙和 单个细胞的膜电流/电势。患者外周血粒细胞 慢性肉芽肿性疾病(CGD)未能显示超氧化物 产生量以及膜电位的变化 激活刺激。我们将确定缺陷是由以下两种原因之一造成的 K+、Cl-或可能的ROCC表达改变。它的外观和 激活后K+通道mRNA的丰度随时间变化的研究 与未激活的巨噬细胞相比。我们将阻止K+通道表达 人K+反义寡核苷酸探针在巨噬细胞中的应用 离子通道表达变化与离子通道表达变化相关的通道克隆 功能上的调制。我们将尝试克隆内心的 巨噬细胞K+通道的整流性及其与细胞功能的关系 巨噬细胞分化。

The interplay of protein phosphorylation, increases in Ca, and changes in membrane potential drives mononuclear phagocyte activation. Ion channel activation in macrophages appears to adjust the gain of the activation signal eliciting the cellular response which includes enhanced secretion, particle phagocytosis, and cytotoxicity. Ion channels may trigger a response by providing a calcium influx pathway, they may modulate a response by altering the driving force governing the influx of calcium, and they may themselves be modulated and/or activated by calcium and phosphorylation. The goal of the proposal is to study multiple signals simultaneously with the goal of determining synergistic interactions that potentiate the phagocyte functional response. We will identify and characterize the voltage-insensitive Ca influx pathway in the phagocytic leukocyte that is opened as a result of agonist binding to surface receptors. We will use fura-2 fluorescence ratio photometry and video-imaging in combination with whole-cell and perforated patch recordings to correlate Cai with membrane current in single cells. Experiments will be performed to determine whether direct activation by G-proteins, phosphorylation, or calcium is involved in the control of receptor-operated Ca2+ permeable channels (ROCCs). It has been suggested that Ca influx through ROCCs is governed by the electrical driving force established by both K+ and Cl- conductances, and therefore we will investigate whether these conductances are tightly coupled to changes in Cai following agonist-stimulation. We will determine whether modulation of membrane K+ and Cl- conductances via neuropeptides and cytokines or lipoxygenase metabolites released by activated macrophages modulate Cai transients in response to surface stimulation. We will use photon counting techniques to follow the respiratory burst activity in single phagocytes activated with both particulate and soluble stimuli. We will establish the correlation between superoxide release, Ca, and membrane current/potential in single cells. Granulocytes from patients with chronic granulomatous disease (CGD) fail to show superoxide production as well as membrane potential changes in response to activating stimuli. We will determine if the defect results from either altered K+, Cl-, or possibly ROCC expression. The appearance and abundance of K+ channel mRNA with time will be studied in activated versus non-activated macrophages. We will block K+ channel expression in macrophages using antisense oligonucleotide probes to human K+ channel clones to correlate alterations in ion channel expression with modulation in function. We will attempt to clone the inwardly rectifying K+ channel in macrophages and examine its relation to macrophage differentiation.