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.
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