Calcium signalling mediated by the α9 acetylcholine receptor in a cochlear cell line from the Immortomouse

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Abstract
We have investigated the characteristics of the α9 acetylcholine receptor (α9AChR) 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 α9 receptor at physiological calcium levels. The data indicate that ACh activates a calcium-permeable channel α9AChR in UB/OC-2 cells and that the channel has a significantly higher calcium permeability than other AChRs. The results indicate that the α9AChR may be able to elevate intracellular calcium levels in hair cells both directly and via store release.