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, 100uM). 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 t1~70 ms and decayed with a bi-exponential time-course ( t2~160, t3~2500 ms) following a 160 ms depolarization to -20mV.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 ~57um2/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.
Dynamics of intracellular calcium in hair cells isolated from the semicircular canal of the frog
RISPOLI, Giorgio;MARTINI, Marta;ROSSI, Marialisa;
2001
Abstract
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, 100uM). 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 t1~70 ms and decayed with a bi-exponential time-course ( t2~160, t3~2500 ms) following a 160 ms depolarization to -20mV.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 ~57um2/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.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
