Ca2+ permeability through rat cloned alpha9-containing nicotinic acetylcholine receptors.

We investigated the functional properties of rat 9 and 910 nicotinic acetylcholine receptors (nAChRs) expressed by transient transfectionin the rat GH4C1 cell line, using both Ca2+ imaging and whole-cell recording. Acute applications of ACh generated short-delay fast-rising and quick-decaying Ca2+ transients, suppressed in Ca2+-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 9 subunit cDNA-transfected GH4C1 cells (9-GH4C1), while they were much larger (range:−150 to−300 pA) in 910 subunit cDNAs-transfected GH4C1 cells (910-GH4C1). Currents were not activated by nicotine, were blocked by methyllycaconitine and were ACh concentration-dependent. Because the Ca2+ permeability of 9-containing nAChRs has been estimated in immortalized cochlear UB/OC-2 mouse cells, we also characterized the ACh-induced responses in these cells. Unlike 9- and 910-GH4C1 cells, UB/OC-2 cells responded to ACh with both long-delay methyllycaconitine-insensitive whole-cell currents and long-lasting Ca2+ transients, the latter being detected in the absence of Ca2+ in the extracellular medium and being suppressed by the Ca2+-ATPase inhibitor thapsigargin, known to deplete IP3-sensitive stores. These results indicated the involvement of muscarinic nAChRs and the lack of functionalACh-gated receptor channels in UB/OC-2 cells. Thus, we measured the fractional Ca2+ current (Pf, i.e. the percentage of total current carried by Ca2+ ions) in 910-GH4C1, obtaining a Pf value of 22±4%; this is the largest value estimated to date for a ligand-gated receptor channel. The physiological role played by Ca2+ entry through 9-containing nAChRs gated by ACh is discussed.