title: ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca2+ signals across the inner ear.
abstract: Extracellular ATP controls various signaling systems including propagation of intercellular Ca(2+) signals (ICS). Connexin hemichannels, P2x7 receptors (P2x7Rs), pannexin channels, anion channels, vesicles, and transporters are putative conduits for ATP release, but their involvement in ICS remains controversial. We investigated ICS in cochlear organotypic cultures, in which ATP acts as an IP(3)-generating agonist and evokes Ca(2+) responses that have been linked to noise-induced hearing loss and development of hair cell-afferent synapses. Focal delivery of ATP or photostimulation with caged IP(3) elicited Ca(2+) responses that spread radially to several orders of unstimulated cells. Furthermore, we recorded robust Ca(2+) signals from an ATP biosensor apposed to supporting cells outside the photostimulated area in WT cultures. ICS propagated normally in cultures lacking either P2x7R or pannexin-1 (Px1), as well as in WT cultures exposed to blockers of anion channels. By contrast, Ca(2+) responses failed to propagate in cultures with defective expression of connexin 26 (Cx26) or Cx30. A companion paper demonstrates that, if expression of either Cx26 or Cx30 is blocked, expression of the other is markedly down-regulated in the outer sulcus. Lanthanum, a connexin hemichannel blocker that does not affect gap junction (GJ) channels when applied extracellularly, limited the propagation of Ca(2+) responses to cells adjacent to the photostimulated area. Our results demonstrate that these connexins play a dual crucial role in inner ear Ca(2+) signaling: as hemichannels, they promote ATP release, sustaining long-range ICS propagation; as GJ channels, they allow diffusion of Ca(2+)-mobilizing second messengers across coupled cells.
authors.affiliation: Foundation for Advanced Biomedical Research, Venetian Institute of Molecular Medicine, 35129 Padua, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Anselmi
authors.forename: Victor H
authors.initials: VH
authors.lastname: Hernandez
authors.forename: Giulia
authors.initials: G
authors.lastname: Crispino
authors.forename: Anke
authors.initials: A
authors.lastname: Seydel
authors.forename: Saida
authors.initials: S
authors.lastname: Ortolano
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Roper
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Kessaris
authors.forename: William
authors.initials: W
authors.lastname: Richardson
authors.forename: Gesa
authors.initials: G
authors.lastname: Rickheit
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Filippov
authors.forename: Hannah
authors.initials: H
authors.lastname: Monyer
authors.forename: Fabio
authors.initials: F
authors.lastname: Mammano
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D000072259:Connexin 26; D000075302:Connexin 30; D017630:Connexins / Q000235:genetics / Q000378:metabolism; D007758:Ear, Inner / Q000166:cytology / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008027:Light; D051379:Mice; D009708:Nucleotidases / Q000378:metabolism; D015290:Second Messenger Systems / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D046509:Tissue Culture Techniques
chemical_list: D002413:Cations, Divalent; D000075302:Connexin 30; D017630:Connexins; D005452:Fluoresceins; C000607042:GJB2 protein, human; C000607043:Gjb2 protein, mouse; C100629:Gjb6 protein, mouse; D000072259:Connexin 26; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D009708:Nucleotidases; D002118:Calcium; C007740:fluorexon