ROLE OF ION CHANNELS IN MONONUCLEAR PHAGOCYTE ACTIVATION

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The engagement of IG Fc receptors on the surface of mononuclear phagocytes triggers a number of cellular responses including particle ingestion, secretion, and respiratory burst activity all of which are integral part of the antimicrobial activity of these cells. The mechanism by which cells within the immune system translate ligand binding to specific membrane receptors into internal messages which direct phagocytosis or secretion remains unclear. It is the aim of this proposal to expand our electrophysiological investigations on the alveolar macrophage, studying the role of both ligand and voltage dependent ion channels in the activation of mononuclear phagocytes using extracellular patch clamp recording techniqus. In this technique, a glass pipette is sealed against a cell allowing one to control the voltage across a small patch of membrane 2-5 Mum2 in area and thereby measure the flow of current through individual membrane channels which open as a result of ligand binding, changes in membrane voltage, or changes in the concentration of an ionic species at one surface of the membrane. We have tentatively identified two populations of voltage dependent K+ selective channels both of which are present in the non-activated macrophage membrane. Experiments will be designed to study the Ca++ dependence of the larger of the two conductance channels in attempt to determine whether this channel is the ubiquitous Ca++ dependent K+ channel and whether the activity of this channel is increased during the phagocytic response. We will study the effects of local anesthetics known to effect both macrophage morphology and activation on K+ channel gating. We will continue our investigations of the IgG-dependent ion channel, which we have characterized in preliminary studies, specifically addressing the question of its Ca++ permeability as well as the Ca++ and voltage dependence of its open state. We will examine the specificity of the reaction using a variety of Fc receptor ligands and the multivalent nature of the response looking at current activation elicited by known oligomeric forms of IgG. We will use the electrical response to IgG as a bioassay to assess the action of Gamma-interferon on monocytes. We will relate current generation to biological response comparing the electrophysiological consequences of agents which induce cellular activation as well as depolarization to those producing only cellular depolarization, using (1) C-reactive protein (2) C3b fragment of complement, (3) f-MLP and analogs and (4) PMA and analogs at both whole cell and single channel level.