GTP-binding (G proteins) proteins are a key link between many cell surface receptors and the cellular response. They act as signal transducers in functions as diverse as control of heart rate and contractility, vision, cell division, control of exocytosis, ribosomal protein synthesis, and olfaction. There are three components of signal transduction in these systems: (1) receptor binding by a specific agonist which catalyzes the (2) splitting of the G protein heterotrimer into alpha-GTP and beta gamma subunits followed by (3) action of the subunits on specific effectors. Examples of effectors are the muscarinic-gated inwardly rectifying potassium (K+) channel in heart, adenylyl cyclase, and phospholipase C. Because there are several types of G protein alpha subunits and fewer (and less well understood) beta gamma subunits, the alpha subunits have been suspected to be the main activation arm of the G protein system. The cardiac muscarinic K+ channel will serve as the assay system to study receptor-G protein and G protein-channel interactions. The following questions will be addressed: (1) What are the G protein subunits that cause the cardiac K+ channel (iK.ACh) to open? We have strong evidence that beta gamma subunits open iK.ACh but we would like to explore the specificity of this action using mammalian heart G proteins on a mammalian atrial membrane. Other alpha and beta gamma subunits from various preparations will be used (including alpha41, alpha40, alpha39) and several tests for specificity of action performed. (2) What are the functional sites of both alpha and beta gamma subunits that allow receptor and/or channel interaction? Chemical modification and antibody binding will be used to modify structure. The functional consequences of these modifications will be compared to controls. (3) Is receptor specificity a function of receptor-G protein interaction? Using a Chinese hamster ovary cell line, four subtypes of muscarinic receptors will be expressed. The link between receptors and ion channels will then be explored using a combination of patch clamp and biochemical techniques. Steps will include characterization of ion currents in CHO cells, search for activation of channels by G protein subunits, and reconstitution of the heterotrimer between receptor and channel. (4) What is the mechanism of desensitization of the K+ current to muscarinic activation? In most cases, inside-out patches of cell membranes will be exposed to intracellular ligands, such as G protein subunits, to determine the steps from receptor binding to channel activation.
