Terrance Egan - US grants

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.

DESCRIPTION (the applicant's description 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.

P2X receptors are transmitter-gated ion channels activated by extracellular ATP. The distribution, topology, pharmacology, and physiology of the seven members of the family (P2X1.7) are well documented. By contrast, the signal transduction pathway is poorly understood. We hypothesize that activation of the receptor involves the following steps: First, ATP binds to a site on the extracellular surface of the protein complex. Second, occupation of this site results in a change in the shape of the channel pore that permits ion conduction to occur. Third, Na[unreadable] and Ca2[unreadable]flow down their electrochemical gradients and into the cell. Fourth, the inward flux of Na* renders the cell hyperexcitable by depolarizing the membrane and the inward flux of Caz* triggers numerous cell-specific sequella such as muscle contraction, neurotransmitter release, and sensation. An additional fiRh step occurs in some receptor subtypes (P2X2,4._)when ATP is applied for more than a few seconds; here, the narrowest part of the pore dilates to a size that allows larger cations like N-methyI-D-glucamine (NMDG) and the cationic cyanine dye, [unreadable]O-PRO-1, to permeate the channel. The functional sequella of dilation include blebbing, microvesiculation, and cell death, actions that may involve intra- and/or inter-molecular interactions of the intracellular C-terminal tail of the receptor. The goal of the experiments outlined in this proposal is to provide a better description of the dynamics of P2X channels during gating, conduction, and pore dilation. In the first aim, we use several techniques to quantify ion flux through homomeric and heteromeric P2X receptors, and we compare these fluxes to those seen in other members of the transmitter-gated ion channel superfamily. Further, we use site-directed mutagenesis to identify domains within the pore that regulate permeability and flux across the surface membrane. In the next two aims, we study the molecular motions of the channel during gating and dilation using two different techniques. In the first set of experiments, an array of cysteine-substituted mutants and thiol-reactive benzophenones will be used to map the position of residues within the transmembrane segments before, during, and after applications of ATP. In the second set of experiments, fluorescence resonance energy transfer (FRET) will be used to determine intra- and inter-molecular distances in the absence and presence of ATP.

[unreadable] DESCRIPTION (provided by applicant): P2X receptors are a family of seven ligand-gated cation channels (LGCCs) that use the energy of ATP binding to initiate a depolarizing flux of cations across cell membranes. Calcium ions carry a disproportionately large percentage of this current, and P2X receptors have one of the largest Ca2+ fluxes of all LGCC families. The resulting rise in intracellular Ca2+ evokes transmitter release from central and peripheral neurons and glia, promotes hormone release from endocrine glands, triggers contraction of muscle, regulates airway ciliary motility, and activates downstream signaling cascades in a variety of cells. ATP has multiple effects on the cardiovascular system. Hematopoietic cells (P2X7), blood vessels (P2XO, and the heart (P2X!.7) all express P2X receptors, and in many cases, the actions of ATP on these tissues are linked to Ca2+ influx. For example, over-expression of the P2X4 receptor enhances cardiac performance and prolongs survival in a transgenic mouse model of heart failure by elevating resting Ca2+ and enhancing basal cardiac contractility. Nature itself may use a similar approach, as P2X receptors are upregulated in the hearts of human patients suffering from dilated cardiomyopathy. Manipulation of endogenous P2X receptors may therefore represent a new therapeutic approach for the treatment of cardiac disease. The focus of our laboratory is the study of the molecular physiology of recombinant P2X receptors. We are particularly interested in describing the mechanics of ATP-gated Ca2+ transport across the membrane and understanding how ATP opens the pore. In this proposal, we outline experiments that build upon our previous work to provide a more quantitative description of the events that follow ATP binding. Relevance to public health: Our work is relevant because it provides the missing information needed to better understand the role that ATP plays in health and sickness. This is important because the receptors may present a new and potentially exciting means of increasing the rate of survival of patients affected by a number of cardiovascular diseases including hypertension and heart failure. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): This application is a request for the 26+ years of continuous support for the Training Program in the Pharmacological Sciences of the Saint Louis University School of Medicine. We seek financial support for six outstanding predoctoral students who will be working for a Ph.D. degree with research emphasis on cellular communication and disease. This is a broadly based, multidisciplinary effort, which involves 21 faculty members from five departments in the Saint Louis University School of Medicine. These departments include: Pharmacological and Physiological Science; Biochemistry and Molecular Biology; Chemistry; Internal Medicine; Otolaryngology and the Center for World Health and Medicine. Students will be selected from candidates who have successfully completed the one year Core Program in Basic Biomedical Science, directly enter with advanced degrees, or M.D./Ph.D. students who have completed the first two years of medical school. We have designed a curriculum that provides in-depth training in Pharmacological Science regardless of the student's background. During the first year of study, all traditional Ph.D. students enroll in the interdisciplinary Core Graduate Program in Biomedical Sciences. This program is designed to provide students with a strong foundation in all aspects of basic biomedical science and the freedom to explore diverse research opportunities. The curriculum combines lectures, small group discussion and seminars. Students entering the Pharmacological Sciences Training Program take advanced pharmacology, journal clubs and seminars. Key to this training program is integration of quantitative pharmacological approaches, application of chemical biology tools, and drug discovery techniques into the formal training program. We utilize several key colleagues from the pharmaceutical industry to enhance this portion of the training program. Subsequent training for all Ph.D. candidates will concentrate on the development of research and teaching competence in a specific area of inquiry under the mentorship of one or more members of the Pharmacological Sciences Training Faculty. The mentors and laboratories participating in this program are well equipped to provide state-of-the-art research training. In addition, core and shared facilities for advanced technologies are available for enhancement of the research training of the participating candidates. Students supported by the T32 Pharmacological Sciences Training Grant have access to a range of unique enrichment activities. The overall objectives of this training program are to provide individuals with the opportunity to achieve a high degree of competence in the area of pharmacological sciences thus preparing them for teaching and research careers.

All P2X receptors transduce significant Ca2+ currents at the resting membrane potential that trigger many of the physiological and pathophysiological actions of extracellular ATP. The molecular physiology of this Ca2+ current is poorly understood despite significant recent advances in functional and structural studies. In this proposal, we focus on the molecular physiology of the Ca2+ current of native and recombinant P2X7 receptors (P2X7Rs). P2X7Rs are prominently expressed in hematopoietic cells and play an essential role in inflammation. They are also found in bone, neurons, and glia where they influence differentiation, homeostasis, and degeneration. Regrettably, meaningful gaps remain in the P2X7R literature despite 40+ years of intensive study. In the experiments outlined in the three specific aims of this proposal, we probe two unknowns. First, we seek to identify the molecular determinates of the greater-than-expected Ca2+ flux of most P2X7Rs. In Specific Aim 1, we use patch-clamp photometry to identify specific domains responsible for the Ca2+ flux. This aim is significant because the structural models derived from crystals of truncated P2XRs may present a distorted view of the permeation pathway and fail to define the ion selectivity filter. In Specific Aim 2, we investigate the curious finding that naturally occurring splice variants of P2X7Rs with distinct N-termini but identical pore-forming helices transduce dramatically different Ca2+ currents, suggesting that the N-termini, which move during gating, play a significant role in positioning an intra-pore Ca2+ selectivity filter. These data leave open the possibility that drugs and signaling complexes that interact with the N-terminus might selectively modulate the physiologically important Ca2+ flux through the channel. Second, we seek to understand the biophysical basis of the time-dependent changes in ATP-current reversal seen during long applications of agonist. Traditionally thought to reflect a gradual dilation of the pore, a recent report suggests that the reversal actually occurs as ions redistribute across the cell surface membrane. In Specific Aim 3, we use novel wild- type and mutant receptors to test the hypothesis that pore dilation and ion accumulation are not mutually exclusive phenomena. These experiments are important because genuine pore dilation could impact Ca2+ homeostasis in living cells. That is, the modified channel structure responsible for dilation could disrupt a key Ca2+ binding site within the P2X7R pore, leading to a reduction in Ca2+ influx and a change in extracellular ATP- dependent cell physiology.