Fine tuning of cardiac function requires a method of controlling heart rate and contractility in a beat-to-beat fashion. Activation of purinergic P2X receptors may accomplish this objective. P2X receptors are a family of ligand-gated ion channels activated by adenosine trisphosphate (ATP). Preliminary evidence suggests that rat heart contains at least 4 Of the 7 members of the P2X receptor family in addition to a P2X receptor in atrial muscle that has yet to be identified at the molecular level. Very little is known about how these receptors work to regulate transmembrane flux of ions. Structural models of P2X receptors share a common motif. Most of the receptor is made of a large hydrophilic extracellular loop that connects two putative transmembrane domains, TM1 and TM2. TM2 is thought to traverse the membrane as an amphipathic alpha-helix and to form a part of the ion channel. The N- and C- termini are thought to be cytoplasmic. To date, no experimental data supporting this model has been presented. The central hypothesis of the proposal outlined in this application is that a more complete characterization of P2X receptors is needed to understand how ATP and the autonomic nervous system control cardiac output. This project outlines experiments that take the first steps towards accomplishing this goal. We are interested in how these proteins regulate the flow of cations across biological membranes. In this regard, the most fundamental unanswered question is what part of the protein forms the ion channel. We hypothesized that one or both of TM 1 and TM2 participates in forming the water-filled ion channel pore. If so, then the similarities and differences in channel function of receptor subtypes may be explained by the patterns of amino acids in these proteins. Our goal is to identify which amino acids line the pore, and to determine how these residues influence conduction. In addition, we expect that a unique sequence of amino acids explains the unusual profile of the atrial P2X receptor. The cDNA encoding this receptor has yet to be identified, and characterization of the properties of the native receptor of atrial muscle is incomplete. We propose to provide a more complete description of the native receptor that takes into account the new information about P2X receptors that has been learned in the last few years.
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