Calcium signalling mediated by the alpha 9 acetylcholine receptor in a cochlear cell line from the Immortomouse

1. We have investigated the characteristics of the alpha 9 acetylcholine re ceptor (alpha 9AChR) expressed in hair cell precursors in an immortalized c ell 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 re cording. 2. Ratiometric measurements of fura-2 fluorescence revealed an increase of intracellular calcium concentration in cells when challenged with 10 mu M A Ch. The calcium increase was seen in 66 % of the cells grown at 39 degrees C in differentiated conditions. A smaller fraction (34 %) of cells grown at 33 degrees C in proliferative conditions responded. 3. 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 mul tiple ACh presentations. Pretreatment with caffeine inhibited all calcium r esponses to ACh. 4. In whole-cell tight-seal recordings 10 mu M ACh activated an inward, non -selective cation current. The reversal potential of the ACh-activated inwa rd current was dependent on the extracellular calcium concentration with an estimated P-Ca/P-Na of 80 for the alpha 9 receptor at physiological calciu m levels. 5. The data indicate that ACh activates a calcium-permeable channel alpha 9 AChR in UB/OC-2 cells and that the channel has a significantly higher calci um permeability than other AChRs. The results indicate that the alpha 9AChR may be able to elevate intracellular calcium levels in hair cells both dir ectly and via store release.