Ca2+ permeability through rat cloned α9-containing nicotinic acetylcholine receptors

We investigated the functional properties of rat alpha 9 and alpha 9 alpha 10 nicotinic acetylcholine receptors (nAChRs) expressed by transient transfection in 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 alpha 9 subunit cDNA-transfected GH4C1 cells (alpha 9-GH4C1), while they were much larger (range: -150 to -300 pA) in alpha 9 alpha 10 subunit cDNAs-transfected GH4C1 cells (alpha 9 alpha 10-GH4C1). Currents were not activated by nicotine, were blocked by methyllycaconitine and were ACh concentration-dependent. Because the Ca2+ permeability of alpha 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 alpha 9- and alpha 9 alpha 10-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 functional ACh-gated receptor channels in UB/OC-2 cells. Thus, we measured the fractional Ca2+ current (P-f, i.e. the percentage of total current carried by Ca2+ ions) in alpha 9 alpha 10-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 Ca2+ entry through alpha 9-containing nAChRs gated by ACh is discussed. (c) 2006 Elsevier Ltd. All rights reserved.