title: Ca2+ permeability through rat cloned alpha9-containing nicotinic acetylcholine receptors.
abstract: We investigated the functional properties of rat alpha9 and alpha9alpha10 nicotinic acetylcholine receptors (nAChRs) expressed by transient transfection in the rat GH4C1 cell line, using both Ca(2+) imaging and whole-cell recording. Acute applications of ACh generated short-delay fast-rising and quick-decaying Ca(2+) transients, suppressed in Ca(2+)-free medium and invariably accompanied by the activation of whole-cell inward currents. The mean amplitude of ACh-induced currents was as small as -16 pA in alpha9 subunit cDNA-transfected GH4C1 cells (alpha9-GH4C1), while they were much larger (range: -150 to -300 pA) in alpha9alpha10 subunit cDNAs-transfected GH4C1 cells (alpha9alpha10-GH4C1). Currents were not activated by nicotine, were blocked by methyllycaconitine and were ACh concentration-dependent. Because the Ca(2+) permeability of alpha9-containing nAChRs has been estimated in immortalized cochlear UB/OC-2 mouse cells, we also characterized the ACh-induced responses in these cells. Unlike alpha9- and alpha9alpha10-GH4C1 cells, UB/OC-2 cells responded to ACh with both long-delay methyllycaconitine-insensitive whole-cell currents and long-lasting Ca(2+) transients, the latter being detected in the absence of Ca(2+) in the extracellular medium and being suppressed by the Ca(2+)-ATPase inhibitor thapsigargin, known to deplete IP(3)-sensitive stores. These results indicated the involvement of muscarinic nAChRs and the lack of functional ACh-gated receptor channels in UB/OC-2 cells. Thus, we measured the fractional Ca(2+) current (P(f), i.e. the percentage of total current carried by Ca(2+) ions) in alpha9alpha10-GH4C1, obtaining a P(f) value of 22 +/- 4%; this is the largest value estimated to date for a ligand-gated receptor channel. The physiological role played by Ca(2+) entry through alpha9-containing nAChRs gated by ACh is discussed.
authors.affiliation: Istituto Pasteur Fondazione Cenci-Bolognetti & Dipartimento di Fisiologia Umana e Farmacologia, Centro di Eccellenza Biologia & Medicina Molecolare, Università di Rome La Sapienza, Italy. sergio.fucile@uniroma1.it
authors.forename: Sergio
authors.initials: S
authors.lastname: Fucile
authors.forename: Antonietta
authors.initials: A
authors.lastname: Sucapane
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Eusebi
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D003001:Cloning, Molecular; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D011978:Receptors, Nicotinic / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
chemical_list: D015220:Calcium Channels; D011978:Receptors, Nicotinic; D019284:Thapsigargin; D000109:Acetylcholine; D002118:Calcium