The aberrant activity of K+ channels and subsequent changes in membrane excitability have been implicated in neurological diseases. G protein-gated inwardly rectifying K+ channels (GIRK) in particular are linked to seizures and neurodegeneration in mice. The long term objective of this research grant is to elucidate the molecular mechanisms underlying G protein activation of GIRK channels. GIRK channels, mostly likely in groups of four subunits (tetramers), are opened during stimulation of G protein-coupled neurotransmitter receptors. While there is general agreement that G protein-derived Gbetagamma subunits activate GIRK channels, and that both the N- and C- termini of GIRK channels bind Gbetagamma, little is known about the physical interaction between Gbetagamma subunits and GIRK channels that governs channel activity. A combination of electrophysiological, molecular genetic, and biochemical techniques will be employed to: (1) Define the functional interactions among the Gbetagamma binding domains in the context of a native GIRK channel. The optimal arrangement of N- and C- termini from GIRK channels will be determined biochemically. The critical number of GIRK subunits and the position of GIRK subunits which donate obligate N- and C-termini for Gbetagamma activation will be determined in genetically engineered GIRK multimers expressed in Xenopus oocytes. (2) Identify the consensus sequences in GIRK channels that are essential for Gbetagamma activation. Gbetagamma activation in genetically altered GIRK channels will be assessed in Xenopus oocytes by monitoring changes in channel activity in the presence of exogenous Gbetagamma subunits. Gbetagamma binding will be measured in affinity-tagged fusion GIRK proteins containing mutant sequences. Gbetagamma channel activity will be tested in the presence of peptide fragments corresponding to regions found to bind Gbetagamma. (3) Identify interacting pairs of amino acids on Gbetagamma subunits and GIRK channels. Potential pairs of interacting amino acids in Gbetagamma and GIRK channels will be identified through biochemical crosslinking techniques. Functional crosslinking of Gbetagamma to GIRK during receptor activation will also be explored. Delineating the signal transduction mechanisms involved in the G-protein activation of these channels may bear directly on design of drug therapies for diseases due to aberrant membrane excitability.
