ROLE OF ION CHANNELS IN MONONUCLEAR PHAGOCYTE ACTIVATION

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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.