VOLTAGE-GATED PROTON CHANNELS IN HUMAN NEUTROPHILS

中性粒细胞在人体的免疫防御系统中起着至关重要的作用。 它们会迁移并杀死细菌。当中性粒细胞通过细胞膜吞噬细菌时，实际的破坏发生。 NADPH氧化酶在静息细胞中无活性，在吞噬体膜中组装并产生活性氧（超氧化物、过氧化氢和次氯酸或漂白剂），这些活性氧直接释放到包围被吞噬细菌的细胞外溶液中，并杀死细菌。 这个过程（“呼吸爆发”）向中性粒细胞释放大量的酸，作为将氧气转化为超氧化物的化学反应的副产品。 持续的超氧化物生产需要从细胞中清除这种酸。 帮助排出这种酸的一种机制是电压门控质子（H+）通道。 电压门控质子选择性离子通道有几个不寻常的特性，使其区别于大多数其他离子通道。 这些特性已经在肺上皮细胞中进行了最彻底的研究，在那里它们可能在CO2消除中发挥作用。 这个项目将描述人类中性粒细胞中H+通道的特性。 将定量测定细胞内和细胞外pH以及膜电位对通道打开和关闭的调节。 电导的温度依赖性，和pH值的依赖性的单个H+通道将被确定通过直接测量（使用低噪声记录技术）或通过噪声分析。 负责上调H+通道功能的活化中性粒细胞的机制将被描绘。 将研究慢性肉芽肿病（CGD）患者的中性粒细胞。 在这种罕见的遗传性疾病中，NADPH氧化酶是有缺陷的，这种细菌杀伤的损害导致严重的感染，通常导致早期死亡。 CGD中的H+电导仅为正常的~ 15%。 我们将确定这种降低H+电导的机制。 H+通道可能是临床干预的靶点，以抵消中性粒细胞的低反应性。

Neutrophils play a vital role in the immunological defense systems of the human body. They migrate toward and kill bacteria. The actual destruction occurs when a neutrophil phagocytoses the bacterium by engulfing it with the cell membrane. The enzyme NADPH oxidase, which is inactive in resting cells, assembles in the phagosome membrane and produces reactive oxygen species (superoxide, hydrogen peroxide, and hypochlorous acid, or bleach) that are released directly into the extracellular solution that surrounds the engulfed bacterium, and which kill the bacteria. This process (the 'respiratory burst') releases into the neutrophil a large amount of acid as a by-product of the chemical reaction that converts oxygen to superoxide. Sustained superoxide production requires removal of this acid from the cell. One mechanism that helps extrude this acid is the voltage-gated proton (H+) channel. Voltage-gated proton-selective ion channels have several unusual properties that distinguish them from most other ion channels. These properties have been studied most thoroughly in lung epithelial cells, where they may play a role in CO2 elimination. This project will characterize the properties of H+ channels in human neutrophils. The regulation of channel opening and closing by intracellular and extracellular pH, and by membrane potential will be determined quantitatively. The conductance temperature dependence, and pH dependence of single H+ channels will be determined either by direct measurements (using low-noise recording techniques) or by noise analysis. The mechanisms responsible for up-regulation of H+ channel function in activated neutrophils will be delineated. Neutrophils from patients with chronic granulomatous disease (CGD) will be studied. In this rare hereditary disease the NADPH oxidase is defective, and this impairment of bacteria-killing results in severe infections often leading to early death. The H+ conductance in CGD is activated only -15 percent of normal. We will determine the mechanism of this reduced H+ conductance. The H+ channel may be a target for clinical intervention to counteract the depressed responsiveness of neutrophils.