title: Knocking out P2X receptors reduces transmitter secretion in taste buds.
abstract: In response to gustatory stimulation, taste bud cells release a transmitter, ATP, that activates P2X2 and P2X3 receptors on gustatory afferent fibers. Taste behavior and gustatory neural responses are largely abolished in mice lacking P2X2 and P2X3 receptors [P2X2 and P2X3 double knock-out (DKO) mice]. The assumption has been that eliminating P2X2 and P2X3 receptors only removes postsynaptic targets but that transmitter secretion in mice is normal. Using functional imaging, ATP biosensor cells, and a cell-free assay for ATP, we tested this assumption. Surprisingly, although gustatory stimulation mobilizes Ca(2+) in taste Receptor (Type II) cells from DKO mice, as from wild-type (WT) mice, taste cells from DKO mice fail to release ATP when stimulated with tastants. ATP release could be elicited by depolarizing DKO Receptor cells with KCl, suggesting that ATP-release machinery remains functional in DKO taste buds. To explore the difference in ATP release across genotypes, we used reverse transcriptase (RT)-PCR, immunostaining, and histochemistry for key proteins underlying ATP secretion and degradation: Pannexin1, TRPM5, and NTPDase2 (ecto-ATPase) are indistinguishable between WT and DKO mice. The ultrastructure of contacts between taste cells and nerve fibers is also normal in the DKO mice. Finally, quantitative RT-PCR show that P2X4 and P2X7, potential modulators of ATP secretion, are similarly expressed in taste buds in WT and DKO taste buds. Importantly, we find that P2X2 is expressed in WT taste buds and appears to function as an autocrine, positive feedback signal to amplify taste-evoked ATP secretion.
authors.affiliation: Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, Florida 33136, USA.
authors.forename: Yijen A
authors.initials: YA
authors.lastname: Huang
authors.forename: Leslie M
authors.initials: LM
authors.lastname: Stone
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Pereira
authors.forename: Ruibiao
authors.initials: R
authors.lastname: Yang
authors.forename: John C
authors.initials: JC
authors.lastname: Kinnamon
authors.forename: Gennady
authors.initials: G
authors.lastname: Dvoryanchikov
authors.forename: Nirupa
authors.initials: N
authors.lastname: Chaudhari
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Finger
authors.forename: Sue C
authors.initials: SC
authors.lastname: Kinnamon
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Roper
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D017630:Connexins / Q000378:metabolism; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D058469:Receptors, Purinergic P2X / Q000096:biosynthesis* / Q000235:genetics; D058476:Receptors, Purinergic P2X2 / Q000096:biosynthesis*; D009435:Synaptic Transmission / Q000235:genetics / Q000502:physiology*; D050053:TRPM Cation Channels / Q000378:metabolism; D013649:Taste / Q000502:physiology; D013650:Taste Buds / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D000098706:Taste Receptors, Type 2
chemical_list: D017630:Connexins; D009419:Nerve Tissue Proteins; C503631:Panx1 protein, mouse; D043562:Receptors, G-Protein-Coupled; D058469:Receptors, Purinergic P2X; D058476:Receptors, Purinergic P2X2; D050053:TRPM Cation Channels; C498617:Trpm5 protein, mouse; D000098706:Taste Receptors, Type 2; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D002118:Calcium