Calcium signalling mediated by the 9 acetylcholine receptor in a cochlear cell line from the Immortomouse.
We have investigated the characteristics of the alpha9 acetylcholine receptor (alpha9AChR) expressed in hair cell precursors in an immortalized cell line UB/OC-2 developed from the organ of Corti of the transgenic H-2Kb-tsA58 mouse (the Immortomouse) using both calcium imaging and whole-cell recording. Ratiometric measurements of fura-2 fluorescence revealed an increase of intracellular calcium concentration in cells when challenged with 10 µM ACh. The calcium increase was seen in 66 % of the cells grown at 39 °C in differentiated conditions. A smaller fraction (34 %) of cells grown at 33 °C in proliferative conditions responded. Caffeine (10 mM) elevated cell calcium. In the absence of caffeine, the majority of imaged cells responded only once to ACh. A small proportion (< 2 % of the total) responded with an increase in intracellular calcium to multiple ACh presentations. Pretreatment with caffeine inhibited all calcium responses to ACh. In whole-cell tight-seal recordings 10 µM ACh activated an inward, non-selective cation current. The reversal potential of the ACh-activated inward current was dependent on the extracellular calcium concentration with an estimated PCa/PNa of 80 for the alpha9 receptor at physiological calcium levels. The data indicate that ACh activates a calcium-permeable channel alpha9AChR in UB/OC-2 cells and that the channel has a significantly higher calcium permeability than other AChRs. The results indicate that the alpha9AChR may be able to elevate intracellular calcium levels in hair cells both directly and via store release.
Jagger
DJ
Department of Physiology, University College London, Gower Street, London WC1E 6BT and Department of Physiology, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.
Griesinger
CB
Rivolta
MN
Holley
MC
Ashmore
JF