The goal is to correlate structure with function for (1) the voltage-gated cardiac Na+ channel and (2) G/K' the regulatory G protein that couples the atrial muscarinic acetylcholine receptor (mAChR) to its ligand-gated inwardly rectifying K+ channel. In this way the molecular bases of channel operation and signal transduction may be explained. The technologies of molecular biology and patch clamp will be used to determine where the structures for voltage gating, ion conductance and tissue specific pharmacology are located in the cardiac Na+ channel and to determine the sites where G/K' interacts with the mAChR and the K+ channel. In addition, the various cardiac G proteins will be cloned and the role of the GTP-ase in the gating mechanism will be studied. In preliminary work oligonucleotide probes to the rat brain Na+ channel(s) cross-hybridized with a rat cardiac cDNA library and were used to isolate recombinant clones as candidates for the rat cardiac Na+ channel gene. Total mRNA from rat heart expressed functional Na+ channels after injection into Xenopus oocytes. Full length cDNA's will be constructed and specific mRNA's will be synthesized in vitro. Chimeric (hybrid) and site-specifically altered cDNA's will be engineered and the corresponding mRNA will be synthesized. The mRNA will be injected for expression into Xenopus oocytes and subsequently studied using patch clamp. In other experiments we proved that cardiac inwardly rectifying K+ channels and mAChR are directly coupled by a G protein, G/K' sensitive to pertussis toxin (PTX) and that cardiac sarcolemma contains at least four PTX substrates separable by electrophoretic techniques under denaturing conditions. Several cDNA's encoding alpha subunits of G proteins have been cloned as well. We now propose to clone cardiac G proteins and identify those unique to heart, as well as G/K'. We will construct chimeric and site specifically mutated G proteins, express them in bacteria and determine their structural- functional parameters. Thus, molecular biology techniques will be used to investigate the structural bases of both voltage gating and ligand gating of ion channels. This project will provide important insights into molecules that are essential for the cardiac impulse. This understanding should be useful in the treatment of cardiac arrhythmias which are the commonest cause of death.
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