title: Imaging P2X4 receptor subcellular distribution, trafficking, and regulation using P2X4-pHluorin.
abstract: P2X4 receptors are adenosine triphosphate (ATP)-gated cation channels present on the plasma membrane (PM) and also within intracellular compartments such as vesicles, vacuoles, lamellar bodies (LBs), and lysosomes. P2X4 receptors in microglia are up-regulated in epilepsy and in neuropathic pain; that is to say, their total and/or PM expression levels increase. However, the mechanisms underlying up-regulation of microglial P2X4 receptors remain unclear, in part because it has not been possible to image P2X4 receptor distribution within, or trafficking between, cellular compartments. Here, we report the generation of pH-sensitive fluorescently tagged P2X4 receptors that permit evaluations of cell surface and total receptor pools. Capitalizing on information gained from zebrafish P2X4.1 crystal structures, we designed a series of mouse P2X4 constructs in which a pH-sensitive green fluorescent protein, superecliptic pHluorin (pHluorin), was inserted into nonconserved regions located within flexible loops of the P2X4 receptor extracellular domain. One of these constructs, in which pHluorin was inserted after lysine 122 (P2X4-pHluorin123), functioned like wild-type P2X4 in terms of its peak ATP-evoked responses, macroscopic kinetics, calcium flux, current-voltage relationship, and sensitivity to ATP. P2X4-pHluorin123 also showed pH-dependent fluorescence changes, and was robustly expressed on the membrane and within intracellular compartments. P2X4-pHluorin123 identified cell surface and intracellular fractions of receptors in HEK-293 cells, hippocampal neurons, C8-B4 microglia, and alveolar type II (ATII) cells. Furthermore, it showed that the subcellular fractions of P2X4-pHluorin123 receptors were cell and compartment specific, for example, being larger in hippocampal neuron somata than in C8-B4 cell somata, and larger in C8-B4 microglial processes than in their somata. In ATII cells, P2X4-pHluorin123 showed that P2X4 receptors were secreted onto the PM when LBs undergo exocytosis. Finally, the use of P2X4-pHluorin123 showed that the modulator ivermectin did not increase the PM fraction of P2X4 receptors and acted allosterically to potentiate P2X4 receptor responses. Collectively, our data suggest that P2X4-pHluorin123 represents a useful optical probe to quantitatively explore P2X4 receptor distribution, trafficking, and up-regulation.
authors.affiliation: Department of Physiology and Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095.
authors.forename: Ji
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Physiology and Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095.
authors.forename: Hua
authors.initials: H
authors.lastname: Chai
authors.affiliation: Institute of General Physiology, University of Ulm, 89081 Ulm, Germany.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Ehinger
authors.affiliation: Department of Pharmacological and Physiological Science and The Center for Excellence in Neuroscience, Saint Louis University School of Medicine, St. Louis, MO 63130 Department of Pharmacological and Physiological Science and The Center for Excellence in Neuroscience, Saint Louis University School of Medicine, St. Louis, MO 63130.
authors.forename: Terrance M
authors.initials: TM
authors.lastname: Egan
authors.affiliation: Department of Physiology and Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095.
authors.forename: Rahul
authors.initials: R
authors.lastname: Srinivasan
authors.affiliation: Institute of General Physiology, University of Ulm, 89081 Ulm, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Frick
authors.affiliation: Department of Physiology and Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095Department of Physiology and Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095 bkhakh@mednet.ucla.edu.
authors.forename: Baljit S
authors.initials: BS
authors.lastname: Khakh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: C485187:PHluorin; D058848:Receptors, Purinergic P2X4; D049452:Green Fluorescent Proteins