An ATP Receptor-Operated ionic conductance in Paramecium

DESCRIPTION (provided by applicant): In vertebrates, ATP is a neurotransmitter, vasodilator and an intercellular signal for stretch and pain. All of these actions can be elicited by activation of specific membrane ATP receptors (P2X receptors) which face the outside of the cell. The genes that code for these P2X receptors produce a gene product that is both the receptor and the ion conducting channel, typically a Ca++ conductance. The pairthat needs to be filled is that very little is known about the genetic and biochemical mechanisms involved in regulating this specialized membrane ion conductance during adaptation to an ATP stimulus. The eukaryotic unicell Paramecium shows ATP-induced behavioral responses (backward swimming), inhibition of these responses by a vertebrate ATP receptor antagonist (PPNDS), ATP-induced depolarizations, high affinity external 3zP-ATP binding and contains membrane proteins that are recognized by peptide antibodies directed to vertebrate P2X1 receptors. My long term objective is to characterize both the ion conductance associated with the AT-induced depolarization of Paramecium and the processes regulating its functional expression during ATP adaptation. To begin this work, my specific aims are to: 1. Characterize the wild type ATP-induced depolarization and currents seen under voltage clamp conditions for their amplitudes, kinetics, ion dependencies and time course of changes during adaptation, 2. Use classical behavioral mutant selection procedures (forward genetics) to obtain behavioral mutants which either don't respond to ATP or don't adapt to ATP, 3. Use "gene silencing" (reverse genetics) to produce genetically altered cell lines that are functional knockouts of the ATP receptor, the ecto-ATPase (which inactivates the ATP signal) and other parts of the signal transduction and adaptation pathway and 4. Use intracellular electrophysiology, in vivo 32p-ATP binding assays and western blot analysis to show whether these genetically-altered cells have normal responses to ATP, external ATP binding and expression of ATP receptors on their plasma membranes. The health-relatedness is that since ATP reception affects neurotransmission, blood flow to kidney and digestive organs, stretch signals coming from hollow organs and pain reception, normal functions of organs of the body can be compromised by alterations in ATP reception. Since Paramecium is the simplest eukaryote to show an ATP receptor that is similar to vertebrate ATP receptors, it offers a unique model system to use both forward and reverse genetic approaches to understand both the excitation and adaptation phases of the ATP response.