TRANSMEMBRANE SIGNALING IN IMMUNOLOGICAL DEGRANULATION

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Secretion of vasoactive amines, SRSA, prostaglandins and other inflammatory mediators is a process which the patient, clinician, pharmaceutical chemist, and basic scientist would each profit from understanding. Systemic anaphylaxis and other manifestations of acute inflammation are a direct result of antigen-induced cross-linkage of immunoglobulin E (IgE) bound to Fc receptors on the surface of mast cells and basophils. The immediate cytoplasmic message created by cross-linked Fc receptors is not known; several biochemical reactions are set in motion, but exactly how they are triggered, and in what way they are coupled to vesicle-membrane fusion remain uncertain. Much evidence indicates that gating of ionic channels in the plasma membrane is an early consequence of antigenic challenge; calcium entry is implicated as an essential step, and membrane depolarization occurs even without calcium influx. So far, however, the evidence for channel activation is either indirect or of limited time-resolution. The goal of this project is to use the powerful technique of gigaseal patch-clamp recording to directly observe the operation of ionic channels in the plasma membrane of degranulating mast cells and rat basophilic leukemia cells. Channel types present in unstimulated cells will first be identified and characterized as to ion selectivity, voltage dependence, open time, unit conductance, and pharmacological profile. The contribution which these channels make to degranulation will then be assessed. Specific questions to be answered are: 1) Is the cross-linked Fc Epsilon receptor an ion channel and, if so, what is its ion specificity? 2) Does bridging of Fc Epsilon receptors generate an intracellular second messenger which gates channel activity in other proteins? 3) How is Ca influx related to membrane depolarization, i.e., does the same or a different channel mediate the two events? 4) Is channel inactivation responsible for the phenomenon of desensitization? 5) Do specific channel blockers prevent exocytosis and, if so, at what points do they interfere with the biochemistry of degranulation? In a related study, the mechanism of electric field-induced serotonin release from rat basophilic leukemia cells will be examined. The long-term objective of this work is to understand the nature of transmembrane signaling devices within the immune system.