The heart beats at an intrinsic rate which is modulated by sympathetic and parasympathetic innervation. The parasympathetic innervation, via the vagus nerve, slows heart rate and modulates contractility by secreting acetylcholine onto muscarinic reports (largely m2 subtype) on pacemaker cells of the heart. The bound muscarinic receptor activates a pertussis toxin-sensitive G protein heterotrimer (G alpha beta gamma) which, in turn, activates an inwardly rectifying K+-selective channel. The steps in G protein subunit(s) activation of the channel, I/KACh, have been the subject of intense investigation, not only because the channel is an important therapeutic target for control of supraventricular arrhythmias, but also because it is a unique model system for understanding G protein- mediated signal transduction. We propose to define the molecular mechanisms underlying G protein regulation of the muscarinicgrated, inwardly-rectifying potassium channel, I/KACh. Recently, two rat atrial cDNAs (GIRK1 and KGA) have been cloned, and when expressed, exhibit many of the properties of I/KACh. Also, only recently have functional recombinant G protein beta-gamma subunits (G beta gamma) been purified and characterized. These two breakthroughs will enable us to systematically investigate the molecular structures regulating I/KACh. The experiments we propose are based on work from our laboratory which indicates that G beta gamma is the physiological mediator of I/KACh activation. There are three specific aims. First we will characterize heterologously- expressed GIRK1 in Xenopus oocytes, Sf9 cells, and GIRK1-transfected Chinese hamster ovary (CHO) cells to lay the groundwork for channel mutagenesis.
Our second aim i s to determine the molecular structures in GIRK1 interacting directly with G beta gamma. We hypothesize a direct interaction between G beta gamma and GIRK1, and based on a comparison of GIRK1 with the G protein-insensitive IRK1, we speculate that the carboxyl-terminal portion of GIRK1 is the G protein regulatory region. We will make chimeras of IRK1 and GIRK1, and express GIRK1 deletion mutants to test this hypothesis. We will express and purify whole channel and cytoplasmic channel fragments from baculoviral and E coli expression systems and use electrophysiological and biochemical approaches to localize the region of GIRK1 that interacts with G beta gamma. Third, we will explore potential effector interaction domains within G beta gamma. The structure-dependent function of G beta gamma is unknown since G beta gamma effectors have only recently been identified. We will modify residues in G gamma to alter its isoprenylation and compare these altered G beta gamma s for their ability to activate I/KACh and GIRK1. Finally, will alter the G beta subunit to test for potential channel binding domains in the G beta region of the molecule.
