A molecular mechanism for parasympathetic nervous modification of cellular excitability involves activation of a muscarinic-gated atrial potassium channel known as GIRK-1. The activation pathway appears to involve a direct interaction of G proteins with the ion channel subunit. The location and the stoichiometry of this interaction remains unknown. Recent evidence proposes that the GIRK-1 subunit coassembles with another inwardly rectifying potassium channel subunit known as CIR. Amino acid deletions in the C-terminus of GIRK render the molecule insensitive to G protein activation. The goal of the experiments in this proposal is to identify the sites in the GIRK protein which affect activation coupling of G proteins to the channel without affecting channel assembly. Channel subunits will be tagged with an epitope to allow specific antibody binding and to permit identification. Experiments will be done to mutate regions of the channel that are predicted to be involved in protein-protein interactions. Mutagenesis experiments will be done to determine whether the channels are functional or not and, if not, it will be determined whether the mutant channels are synthesized and targeted to the plasma membrane by using the epitope tagged subunits. Secondly, it will be determined whether these regions that are critical to the function of the GIRK channel are also G protein binding sites. The third strategy will be to try and revert nonfunctional mutant GIRK-1 channels by creating a chimeric construct containing structural elements known to bind G proteins from other systems. These investigations will provide evidence to help identify the structural elements involved in coupling between G proteins and this important class of inwardly rectifying potassium channels. In a broader sense, the high resolution measurements achievable in these types of experiments may provide important insight into our understanding of how G proteins interact with other types of proteins to which they are known to couple. Molecular information about G protein interactions and about this class of ion channels is important for understanding the molecular basis of excitability, cellular signalling and information transfer.
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