Dynamics of intracellular calcium in hair cells isolated from the semicircular canal of the frog

Changes in cytosolic free Ca2+ concentration ([Ca2+](i)) were monitored optically in hair cells mechanically isolated from frog semicircular canals using the membrane-impermeant form of the Ca2+-selective dye Oregon Green 488 BAPTA-1 (OG, 100 muM). Cells stimulated by depolarization under whole-cell voltage clamp conditions revealed Ca2+ entry at selected sites (hotspots) located mostly in the lower (synaptic) half of the cell body. [Ca2+](i) at individual hotspots rose with a time constant tau (1)similar to 70 ms and decayed with a bi-exponential time-course ( tau (2)similar to 160, tau (3)similar to 2500 ms) following a 160 ms depolarization to - 20 mV. With repeated stimulation [Ca2+](i) underwent independent amplitude changes at distinct hotspots, suggesting that the underlying Ca2+ channel clusters can be regulated differentially by intracellular signalling pathways. Block by nifedipine indicated that the L-type Ca2+ channels are distributed at different densities in distinct hotspots. No diffusion barrier other than the nuclear region was found in the cytosol, so that, during a prolonged depolarization (lasting up to 1 s), Ca2+ was able to reach the cell apical ciliated pole. The effective Ca2+ diffusion constant, measured from the progression of Ca2+ wavefronts in the cytosol, was similar to 57 mum(2)/s, Our results indicate that in these hair cells, buffered diffusion of Ca2+ proceeds evenly from the source point to the cell interior and is dominated by the diffusion constant of the endogenous mobile buffers. (C) 2001 Harcourt Publishers Ltd.