Voltage-Gated Proton Channels in Human Neutrophils

描述（由申请人提供）：该项目的目标是了解人类中性粒细胞和相关细胞杀死细菌和其他微生物入侵者的机制。2个重要的分子实体，酶，NADPH氧化酶和电压门控质子通道是本项目的重点。NADPH氧化酶产生超氧阴离子，这是杀死微生物的活性氧的前体。这种酶的工作原理是在细胞膜上传递电子，从而产生电流，这种电流将通过“穿孔贴片”和“切除贴片”电压钳技术进行测量。正如最近所证明的那样（DeCoursey et al, 2003），如果没有电荷补偿机制，由这种电流引起的去极化几乎会立即停止酶的功能。假设（Hendeson et al ., 1987）电荷补偿是通过质子通量通过通道实现的。质子通道介导电流将使用穿孔贴片技术进行研究。这种方法可以同时研究NADPH氧化酶和质子通道在活细胞中的功能，目前只有我们的实验室使用。(1)我们将确定NADPH氧化酶的电压、温度和底物依赖性。(2)我们将通过研究吞噬细胞（中性粒细胞和嗜酸性粒细胞）中超氧化物的产生、用Zn2+抑制质子通道和使用离子载体来评估质子通道在电荷补偿中的作用。我们将研究最近的一项建议，即K通道在电荷补偿和微生物杀灭中发挥重要作用（Ahluwalia et al, 2004）。(3)我们将确定NADPH氧化酶功能对pHo和pHi的依赖性。在我们的假设中，NADPH氧化酶与质子通道的通讯机制之一是细胞内ph。(4)将探讨NADPH氧化酶与质子通道的协调调节。将研究几种生理NADPH氧化酶激动剂，以确定它们对氧化酶和质子通道的影响。我们将抑制信号通路，以确定质子通道功能是如何调节的。人类吞噬细胞在杀死细菌时设法打开NADPH氧化酶和质子通道-该项目将阐明这两个关键分子是如何协调调节的。(5)确定质子通道分子和基因。

DESCRIPTION (provided by applicant): The goal of this project is to understand the mechanisms involved in the killing of bacteria and other microbial invaders by human neutrophils and related cells. 2 molecular entities of central importance, the enzyme, NADPH oxidase and voltage-gated proton channels are the focus of this project. NADPH oxidase produces superoxide anion, the precursor to reactive oxygen species that kill microbes. This enzyme works by transporting electrons across the cell membrane, which generates an electrical current that will be measured using the "perforated-patch" and excised-patch voltage-clamp techniques. As demonstrated recently, (DeCoursey et al, 2003) the depolarization caused by this electron current would stop enzyme function almost immediately, if there were no charge compensation mechanism. As hypothesized, (Hendeson et al, 1987) charge compensation is achieved by proton flux through channels. Proton channel-mediated currents will be studied using the perforated-patch technique. This approach, which allows simultaneous study of both NADPH oxidase and proton channel function in living, responsive cells, is currently used only by our lab. (1) We will determine the voltage-, temperature-, and substrate-dependence of NADPH oxidase. (2) We will evaluate the proposed role of proton channels in charge compensation, by studying superoxide production in phagocytes (neutrophils and eosinophils), inhibiting proton channels with Zn2+, and using ionophores. We will examine a recent proposal that K channels play important roles in charge compensation and microbe killing (Ahluwalia et al, 2004). (3) We will determine the dependence of NADPH oxidase function on pHo and pHi. In our hypothesis, 1 mechanism of communication between NADPH oxidase and proton channels is intracellular pH. (4) The coordinated regulation of NADPH oxidase and proton channels will be explored. Several physiological NADPH oxidase agonists will be studied to determine their effects on both the oxidase and proton channels. We will inhibit signaling pathways to determine how proton channel function is regulated. Human phagocytes manage to turn on NADPH oxidase and proton channels when they kill bacteria - this project will clarify how the coordinated regulation of these 2 key molecules is accomplished. (5) We will identify the proton channel molecule and gene.