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

1. 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. 2. Ratiometric measurements of fura-2 fluorescence revealed an increase of intracellular calcium concentration in cells when challenged with 10 microM ACh. The calcium increase was seen in 66 % of the cells grown at 39 degrees C in differentiated conditions. A sm aller fraction (34%) of cells grown at 33 degrees C in proliferative con ditions responded. 3. Caffeine (10mM) elevated cell calcium. In the ab sence of caffeine, the majority of imaged cells responded only once to A Ch presentations. Pretreatment with caffeine ingibited all calcium respo nses to ACh. 4. In whole-cell tight-seal recordings 10 microM ACh activa ted inward current was dependent on the extracellular calcium concentrat ion with an estimated PCa/PNa of 80 for the alpha9 receptor at physiological calcium levels. 5 . The data indicate that ACh activates a calcium-permeable channel alpha 9AChR in UB/OC-2 cells and that the channel has a significantly higher c alcium permeability than other AChRs. The results indicate that the alp ha9AChR may be able to elevate intracellular calcium levels in hair cell s both directly and via store release.