Confocal imaging of calcium microdomains and calcium extrusion in turtle hair cells

We have studied spatial Ca2+ distribution in hair cells filled with the low affinity fluorescent indicator Calcium Green 5N using real-time confocal microscopy and whole-cell recording. During depolarizations lasting several hundred milliseconds, Ca2+ fluorescence increased at a number of hotspots around the base of the cell but changed little near the hair bundle. The hotspots required influx of Ca2+ through voltage-dependent channels, and they expanded during the pulse from an initial diameter of < 1 micron. Strong Ca2+ buffers like BAPTA slowed their growth rate. On repolarization, the fluorescence decayed with two time constants: approximately 0.1 s, which may represent Ca2+ diffusion away from the entry sites, and 10 s, probably reflecting Ca2+ extrusion. Extrusion occurs mainly via a CaATPase that can be blocked by vanadate. We suggest the hotspots are microdomains of Ca2+ attaining a concentration of at least 85 microM near assemblies of synaptic release sites.