Acetylcholine, a neurotransmitter implicated in memory and Alzheimer's disease, binds to and activates ligand-gated ion channels, G protein Gq-coupled receptors, and G protein Gi-coupled receptors. Hence, a single neurotransmitter can access a multitude of signaling pathways in neurons. Because activation of each of these pathways could result in a different biological response, neurons must functionally segregate these different signaling pathways. The molecular mechanism by which neurons isolate signaling pathways remains an enigma and is one of the most important questions facing Neuroscientists today. The study of G protein-gated inwardly rectifying K+ channels (GIRK) is an excellent system for identifying the molecular determinants of receptor specificity. GIRK channels are opened by the G protein G beta gamma subunits but show little discrimination among the different combinations of G beta and Ggamma, subunits in vitro. Because specific combinations of G beta gamma subunits do not selectively open GIRK channels, any G protein-coupled neurotransmitter receptor could theoretically open GIRK channels. However, neurons ensure that acetylcholine will open GIRK channels following stimulation of G protein Gi-coupled but not Gq-coupled cholinergic receptors. This type of receptor specificity is common among many different types of neurotransmitter signaling pathways. In this proposal, the hypothesis that a protein-protein interaction between the GIRK channel and a subset of G proteins determines the specificity of receptor coupling will be examined. The hypothesis will be tested using biochemistry for examining direct protein-protein binding interactions, electrophysiology for assessing the physiology of GIRK channels, laser confocal microscopy for visualizing the localization of GIRK channels and neurotransmitter receptors, and finally fluorescence spectroscopy for measuring protein-protein interactions in living cells. In addition, we will be using cultures of hippocampal neurons to identify the domains in GIRK channels that are important for targeting to the dendritic shafts. Results from this grant will significantly advance our understanding of the principles governing coupling of G protein-coupled receptors to GIRK channels, and may contribute to specific pharmaceutical strategies for treating humans with diseases that are caused by abnormal neuronal membrane excitability.