verbatim): Adenosine triphosphate (ATP) is unusual in its ability to influence cell activity from both the intracellular and extracellular compartments. Intracellular hydrolysis of ATP to adenosine 5'-diphosphate and inorganic phosphate provides the energy needed to power a range of energetically unfavorable chemical reactions and is an important source of phosphate in many biosynthetic reactions. Extracellular ATP modulates cell excitability by activating membrane-bound P2 purinoceptors. One branch of this family, the P2X receptors, are themselves a class of ligand-gated ionic channels that conduct the flow of cations across the cell surface membranes of a variety of tissue. Cationic conduction occurs when the integrant ion channel opens as a result of agonist occupation of an extracellular binding site. However, the molecular mechanisms of gating and selectivity remain a mystery due in part to an incomplete mapping of the functional domains of the receptor-channel complex. Further, the role these proteins play in control of cell excitability of native tissues is poorly defined. The experiments outlined in this proposal are designed to address these deficiencies using both recombinant purinergic receptors expressed in human embryonic kidney cells and native receptors of acutely-dissociated rat atrial muscle. The first specific aim is to map the relationship between structure and function of the P2X2 receptor. We propose to identify amino acid that contribute to the selectivity filter and the channel gate using an inclusive site-directed mutagenesis approach that considers both putative pore-forming domains. Our preliminary data demonstrate this approach will be successful. The second specific aim explores the contribution of these domains to the newly discovered """"""""dilated"""""""" mode of the channel pore. We hypothesize that the large cations that permeate the dilated channel traverse the same permeation pathway used by small mono- and divalent cations in the """"""""constricted"""""""" pore mode. Again, site-directed mutagenesis will be used to identify differences in the secondary and tertiary structure of the dilated and constricted channels. The third specific aim is to characterize the native purinergic current of rat atrial muscle. Although ATP is known to depolarize frog atrial muscle by activating an unknown member of the P2X receptor family, little is known about the effect of ATP on mammalian atria. Our preliminary data strongly suggest that rat atria express a functional P2X receptor that is unique. A more complete characterization of the physiology and pharmacology of this receptor will help define the role of ATP in the pharmacology, physiology, and pathophysiology o mammalian cardiac muscle.
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