Changes in cytosolic free Ca2+ concentration ([Ca2+]i) were rnonitored optically from hair cells mechanically isolated from frog sernicircular canals. Cells stimulated by depolarization under whole-cell voltage clamp were imaged at 4.1 ms inter-frame intervals, revealing Ca2+ entry at seleoted sites (hotspots) located at the synaptic pole. To correlate [Ca2+]i changes with release of tranamitter at the cytoneural junction, miniature excitatory postsynaptic potentials (rnEPSFs) were also recorded from afferent nerve fibers in an intact labyrinth preparation subjected to angular accelerations. In isolated cells, [Ca2+]i at individual hotspots rose with a time constant tau1 of about 70 ms and decayed with a bi-exponential time-course (tau2 of about 160, and tau3 of about 2500 ms) following brief depolarizations. With repeated stimulation [Ca2+]i underwent independent amplitude changes at distinct hotspots, suggesting that the underlyirtg voltage-operated Ca channel (VOCC) clusters can be regulated differentially by intracellular signaling pathways. Furthermore, block by nifedipine indicated that the L-type VOCCs are distributed at different densities in distinct hotspots. The apparent Ca2+ diffusion constant in the cytosol resulted aboutt 57 microm2/s. Analysis of diffusion patterns showed that Ca2+ reached the cell apical membrane during 0.15-1.0 s depolarizations. Recordings of rnEPSPs showed that depolarizations of comparable duration could be elicited in the intact labyrinth either by long-lasting mild acceIerations or by large angular velocity steps. It is proposed that, during sustained activation of VOCCs, Ca2+ diffusing up to the hair bundle might interfere with the adaptation of the transduction current and associated bundle rnovements.
Ca2+ DYNAMICS IN FROG SEMICIRCULAR CANAL HAIR CELLS PROBED BY FAST FLUORESCENCE
RISPOLI, Giorgio;MARTINI, Marta;ROSSI, Marialisa;
2001
Abstract
Changes in cytosolic free Ca2+ concentration ([Ca2+]i) were rnonitored optically from hair cells mechanically isolated from frog sernicircular canals. Cells stimulated by depolarization under whole-cell voltage clamp were imaged at 4.1 ms inter-frame intervals, revealing Ca2+ entry at seleoted sites (hotspots) located at the synaptic pole. To correlate [Ca2+]i changes with release of tranamitter at the cytoneural junction, miniature excitatory postsynaptic potentials (rnEPSFs) were also recorded from afferent nerve fibers in an intact labyrinth preparation subjected to angular accelerations. In isolated cells, [Ca2+]i at individual hotspots rose with a time constant tau1 of about 70 ms and decayed with a bi-exponential time-course (tau2 of about 160, and tau3 of about 2500 ms) following brief depolarizations. With repeated stimulation [Ca2+]i underwent independent amplitude changes at distinct hotspots, suggesting that the underlyirtg voltage-operated Ca channel (VOCC) clusters can be regulated differentially by intracellular signaling pathways. Furthermore, block by nifedipine indicated that the L-type VOCCs are distributed at different densities in distinct hotspots. The apparent Ca2+ diffusion constant in the cytosol resulted aboutt 57 microm2/s. Analysis of diffusion patterns showed that Ca2+ reached the cell apical membrane during 0.15-1.0 s depolarizations. Recordings of rnEPSPs showed that depolarizations of comparable duration could be elicited in the intact labyrinth either by long-lasting mild acceIerations or by large angular velocity steps. It is proposed that, during sustained activation of VOCCs, Ca2+ diffusing up to the hair bundle might interfere with the adaptation of the transduction current and associated bundle rnovements.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
