This is a renewal application of an ongoing effort to determine the role that ion channels play in macrophage function, specifically particle release and subsequent release of inflammatory cytokines. By means of electrophysiology, including single-channel, whole-cell, and capacitance measurements, microfluorimetry and molecular biology, the investigator proposes the following specific aims. First, ion channels activated as a consequence of free radical release following particle uptake in mononuclear phagocytes will be identified. Published and preliminary work from this laboratory have identified a superoxide-generated nonselective, depolarizing membrane current. Electrophysiologic studies of selectivity, gating and pharmacology will be used to identify the channel or channels that produce the free radical-induced current. Second, the relationship between membrane capacitance, ion channel activation, changes in intracellular Ca and intracellular pH following particle uptake will be determined. Phagocytosis and exocytosis will be assayed directly as changes in membrane capacitance. The endocytic event will be correlated with changes in Ca, changes in conductance, and changes in intracellular pH in both primary human-derived macrophage cell lines and a macrophage-like transformed cell line. The goal will be to determine intervention points at which phagocytosis could be uncoupled from secretion. The third and final specific aim is directed toward the determination of the role that the inwardly rectifying K+ channel IRK1 plays in cellular function. The investigator hypothesizes that respiratory burst response as well as pro-inflammatory cytokine secretion will be down-regulated in cells that undergo chronic depolarization as a consequence of a loss of IRK1 expression or function. Investigation of this hypothesis will be accomplished through several approaches. The effect of IRK1 inactivation by Ba2+ (50 - 250 micromolar) on respiratory burst activity in the J774.1 cell line will be determined. Dominant negative, nonfunctional pore mutants if IRK1 will be injected into macrophage cells (both cultured primary and transformed cell lines) to examine the effect on function in single cells. Stable J774.1 cell lines in which IRK1 is knocked out or the outwardly rectifying Kv1.5 is overexpressed by means of a tetracyline-regulated retroviral vector system, will be used to assess the effects of IRK1 expression on later stages of macrophage function, including granule release and gene activation. Finally, transgenes individually containing a genetically designed dominant negative IRK1 mutant will be constructed. A macrophage specific promoter will be used to direct expression to the granulocyte cell population in transgenic mice, in which the role of IRK1 will be assessed.
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