title: Rab5 regulates internalisation of P2X4 receptors and potentiation by ivermectin.
abstract: The P2X4 receptor is an ATP-gated ion channel expressed in neurons, endothelia and immune cells. Plasma membrane expression of P2X4 is regulated by dynamin-dependent endocytosis, and this study identifies a Rab5-dependent pathway of receptor internalisation. Expression of Rab5 constructs altered the distribution of P2X4 in HEK-293 cells, and both constitutive internalisation and agonist-induced desensitisation of P2X4 were increased by co-expression of wild-type Rab5 or constitutively active Rab5 (Q79L). Expression of inactive dynamin K44A and Rab5 S34N constructs abolished agonist-induced desensitisation, suggesting internalisation as the underlying mechanism. Blocking P2X4 internalisation in this way also abolished potentiation of ATP-induced currents by the allosteric modulator ivermectin. This suggests that the dynamin-Rab5 internalisation pathway is essential for the ivermectin potentiation effect. In agreement with this hypothesis, the co-expression of wild-type dynamin, wild-type Rab5 or active Rab5 (Q79L) could increase the potentiation of the ATP-induced P2X4 response by ivermectin. These findings highlight Rab5 GTPase as a key regulator of P2X4 receptor cell surface expression and internalisation.
authors.affiliation: Department of Biomedical Science, University of Sheffield, Sheffield, UK. leanne.stokes@sydney.edu.au
authors.forename: Leanne
authors.initials: L
authors.lastname: Stokes
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D034281:Dynamins / Q000235:genetics / Q000502:physiology; D055724:Electrophysiological Phenomena; D004705:Endocytosis / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D057809:HEK293 Cells; D006801:Humans; D007559:Ivermectin / Q000494:pharmacology*; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques; D058911:Purinergic P2X Receptor Agonists / Q000494:pharmacology; D051381:Rats; D058848:Receptors, Purinergic P2X4 / Q000187:drug effects* / Q000235:genetics / Q000502:physiology*; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: D058911:Purinergic P2X Receptor Agonists; D058848:Receptors, Purinergic P2X4; D007559:Ivermectin; D000255:Adenosine Triphosphate; D020696:rab5 GTP-Binding Proteins; D034281:Dynamins