Ca2+ permeability through rat cloned alpha9-containing nicotinic acetylcholine receptors.
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.
Fucile
Sergio
S
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
Sucapane
Antonietta
A
Eusebi
Fabrizio
F
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
D015220:Calcium Channels; D011978:Receptors, Nicotinic; D019284:Thapsigargin; D000109:Acetylcholine; D002118:Calcium