Identification And Functional Study Of F2X₃Receptor Intracellular Interacting Molecules

P2X₃receptors belong to the ligand-gated ion channels, with intracellular cytoplasmicN and C termini and two transmembrane domains connected by a large extracellular loop.Once ATP binds, they result in a non-selective cationic influx, permeable to sodium,calcium and potassium. P2X₃receptors are expressed predominately on small-andmedium-afferent neurons in most tissues like skin, joints, and hollow organs. Theyparticipate in many crucial physiological processes such as nociceptive transmission,chemical signaling, gastrointestinal peristalsis and bladder reflex. Thus it is of significance toinvestigate the regulation of P2X₃receptor of its expression and function. Much of workhas been done focusing on the pharmacology of the receptor; still many research revealvarious interactions between P2X₃receptor and molecules on the plasma membrane,however, the intracellular modulation and interaction of P2X₃receptor remains poorlyunderstood.To investigate the intracellular regulation of P2X₃receptor, we performed the yeasttwo-hybrid screen, using the59amino acids of the C termini of P2X₃receptor as the baitprotein. We identified6candidates as potential P2X₃receptor intracellular interactingmolecules, and one of them is hepatocyte growth factor-regulated tyrosine kinase substrate（HGS）.HGS is a115KDa protein cloned first in1995. It possesses several functional domains,docks in early endosome membrane and participates in multiple biochemical process i.g.intracellular signal transduction、ubiquitin-dependent protein degradation and membraneprotein recycling. It has been reported as highly enriched in brain and regulatesCa2+-dependent exocytosis. Its expression and function in DRG has not been reported.In the present study, we demonstrate that HGS interacts with P2X₃receptor in vitro.We report that HGS co-localizes well with P2X₃receptor in DRG neurons. Besides, HGSinhibits α, β-me-ATP induced P2X₃receptor current when both expressed in HEK293Tcells.