title: Identification of human P2X1 receptor-interacting proteins reveals a role of the cytoskeleton in receptor regulation.
abstract: P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation.
authors.affiliation: Department of Cell Physiology and Pharmacology, Henry Wellcome Building, University of Leicester, Leicester LE1 9HN, United Kingdom.
authors.forename: Ulyana
authors.initials: U
authors.lastname: Lalo
authors.affiliation: Department of Cell Physiology and Pharmacology, Henry Wellcome Building, University of Leicester, Leicester LE1 9HN, United Kingdom.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Roberts
authors.affiliation: Department of Cell Physiology and Pharmacology, Henry Wellcome Building, University of Leicester, Leicester LE1 9HN, United Kingdom. Electronic address: rje6@le.ac.uk.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Evans
mesh_terms: D000199:Actins / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D015638:Cytochalasin D / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D047630:Depsipeptides / Q000494:pharmacology; D004594:Electrophysiology / Q000379:methods; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods; D008954:Models, Biological; D032389:Myocytes, Smooth Muscle / Q000166:cytology; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D058474:Receptors, Purinergic P2X1 / Q000378:metabolism*
chemical_list: D000199:Actins; D047630:Depsipeptides; D058474:Receptors, Purinergic P2X1; C057531:jasplakinolide; D015638:Cytochalasin D; D000255:Adenosine Triphosphate; D011493:Protein Kinase C