ROLE OF ION CHANNELS IN MONONUCLEAR PHAGOCYTE ACTIVATION

蛋白质磷酸化的相互作用，钙的增加， 在膜电位驱动单核吞噬细胞活化。 离子 巨噬细胞中的通道激活似乎可以调节 激活信号引发细胞应答，所述细胞应答包括 增强的分泌、颗粒吞噬作用和细胞毒性。 离子 通道可通过提供钙内流途径触发反应， 它们可以通过改变控制反应的驱动力来调节反应 钙的内流，并且它们本身可以被调节和/或激活 通过钙和磷酸化。 该提案的目的是研究 多个信号同时进行，目的是确定协同 增强吞噬细胞功能反应的相互作用。 我们将 识别和表征电压不敏感的Ca内流途径， 由于激动剂结合而打开的吞噬性白细胞 到表面受体。 我们将使用Fura-2荧光比率光度法 和视频成像结合全细胞和穿孔贴片 记录以将Cai与单细胞中的膜电流相关联。 将进行实验以确定是否通过 G蛋白、磷酸化或钙参与了 受体操纵的Ca 2+渗透通道（ROCC）。 已经 表明，钙内流通过ROCCs是由电 由K+和Cl-电导建立的驱动力，因此 我们将研究这些电导是否与 激动剂刺激后Cai的变化。 我们将决定 通过神经肽调节膜K+和Cl-电导， 由活化的巨噬细胞释放的细胞因子或脂氧合酶代谢物 响应于表面刺激调节Cai瞬变。 我们将使用 光子计数技术跟踪呼吸爆发活动， 用颗粒和可溶性刺激物激活的单个吞噬细胞。 我们将建立超氧化物释放，钙， 单细胞膜电流/电位。 患者粒细胞 患有慢性肉芽肿病（CGD）的患者未能显示超氧化物 生产以及膜电位的变化， 激活刺激。 我们将确定缺陷是否由以下原因造成： 改变K+、Cl-或可能的ROCC表达。 外观和 K+通道mRNA丰度随时间的变化将在活化的细胞中进行研究。 与未激活的巨噬细胞相比。 我们将阻断K+通道表达 在巨噬细胞中使用针对人K+的反义寡核苷酸探针 通道克隆，以将离子通道表达的改变与 功能上的调制 我们将尝试克隆 纠正巨噬细胞中的K+通道，并检查其与 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.