The described project continues to examine control of ion channels by intracellular pathways. Experiments will be conducted to understand the molecular and mechanistic details of the activation pathway leading from the 7-helix receptors to G-proteins and to the inwardly rectifying KGA potassium channel. Problems to be addressed include: the identity of the G-protein subunits involved, the nature of activation by Gs-coupled receptors; the relationship between permeation, magnesium block and slow gating; and stoichiometry of the assembled channel. Using the oocyte preparation, methods will be developed and exploited for controlling the cytoplasmic surface (perfused and injected oocytes) and for single channel (excised inside-out patches) experiments. Mammalian systems will be developed for heterologous expression of KGA. Biochemical experiments will study direct binding of labelled tracer amounts of appropriate segments of KGA (including intracellular regions produced by in vitro translation) with biochemical quantities of G-protein subunits attached to solid supports in order to identify regions on KGA and the specified G-protein subunits that interact. GTPase activating characteristics of the KGA channel will be addressed, as will the identity of the inwardly rectifying K-channel that is inhibited by a G-protein pathway in oocytes expressing rat brain mRNA. The applicant indicates that the channel to be studied is important in the control of heartbeat, analgesia, and in drug abuse.
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