K and Ca ions play an essential role in hair cell transduction mechanisms. K carries the transduction current at the apical ciliated pole of the cell, where localised cytosolic free Ca concentration ([Ca ]i) changes modulate the open probability of the mechanosensitive channels and thus regulate the amplitude of the receptor potential. This, in turn, activates Ca and K basolateral conductances, which ultimately control transmitter release at the cell's cytoneural junction. Some vertebrate hair cells display cytosolic microdomains where the [Ca]i increases markedly in response to depolarisation. These Ca hotspots are thought to depend on focal Ca entry through clusters of voltage-activated Ca channels colocalised with Ca -activated K channels at presynaptic active zones. Ca microdomains have been studied in hair cells of the frog sacculus and in auditory hair cells of the turtle, but no studies of Ca dynamics have been performed on hair cells of the semicircular canal. To further characterise the spatial and temporal features of the [Ca]i changes associated to the gating of voltage-activated Ca channels, we have applied fast CCD imaging of intracellular Ca and patch clamp recording to hair cells from the crista arnpullaris of Rana esculenta. Changes in [Ca]i were optically monitored using the membrane-impermeant form of the Ca-selective dye Oregon Green 488 BAPTA-1. Cells were stimulated by depolarizing voltage commands during whole cell voltage clamp recordings and concurrently imaged at 247 Hz frame rate. Cells stimulated by depolarisation revealed Ca entry at selected sites (hotspots) located mostly in the lower (synaptic) half of the celi body. [Ca]i at individual hotspots rose with a time constant of about 70 ms and decayed with a bi-exponential time-course (time constants: 160 and 2500 ms) following a 160 ms depolarisation to -20 mV. With repeated stimulation [Ca]i underwent independent amplitude changes at distinct hotspots, suggesting that the underlying Ca channel clusters can be regulated differentially by intracellular signalling pathways. Block by nifedipine indicated that the L-type Ca channels are distributed at different densities in distinct hotspots. The study of diffusion away from the hotspot indicates that no major diffusional barriera other than the nuclear region exists in the cytoplasm, and the Ca clearance machinery is also likely clustered at selected locations. Therefore, during a prolonged depolarisation (lasting up to 1 s), Ca was able to reach the cell apical ciliated pole. The effective Ca diffusion constant, measured from the progression of Ca wavefronts in the cytosol, was about 57 micron2/s. Our results indicate that, in these hair cells, buffered diffusion of Ca 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 frog
RISPOLI, Giorgio;MARTINI, Marta;ROSSI, Marialisa;
2001
Abstract
K and Ca ions play an essential role in hair cell transduction mechanisms. K carries the transduction current at the apical ciliated pole of the cell, where localised cytosolic free Ca concentration ([Ca ]i) changes modulate the open probability of the mechanosensitive channels and thus regulate the amplitude of the receptor potential. This, in turn, activates Ca and K basolateral conductances, which ultimately control transmitter release at the cell's cytoneural junction. Some vertebrate hair cells display cytosolic microdomains where the [Ca]i increases markedly in response to depolarisation. These Ca hotspots are thought to depend on focal Ca entry through clusters of voltage-activated Ca channels colocalised with Ca -activated K channels at presynaptic active zones. Ca microdomains have been studied in hair cells of the frog sacculus and in auditory hair cells of the turtle, but no studies of Ca dynamics have been performed on hair cells of the semicircular canal. To further characterise the spatial and temporal features of the [Ca]i changes associated to the gating of voltage-activated Ca channels, we have applied fast CCD imaging of intracellular Ca and patch clamp recording to hair cells from the crista arnpullaris of Rana esculenta. Changes in [Ca]i were optically monitored using the membrane-impermeant form of the Ca-selective dye Oregon Green 488 BAPTA-1. Cells were stimulated by depolarizing voltage commands during whole cell voltage clamp recordings and concurrently imaged at 247 Hz frame rate. Cells stimulated by depolarisation revealed Ca entry at selected sites (hotspots) located mostly in the lower (synaptic) half of the celi body. [Ca]i at individual hotspots rose with a time constant of about 70 ms and decayed with a bi-exponential time-course (time constants: 160 and 2500 ms) following a 160 ms depolarisation to -20 mV. With repeated stimulation [Ca]i underwent independent amplitude changes at distinct hotspots, suggesting that the underlying Ca channel clusters can be regulated differentially by intracellular signalling pathways. Block by nifedipine indicated that the L-type Ca channels are distributed at different densities in distinct hotspots. The study of diffusion away from the hotspot indicates that no major diffusional barriera other than the nuclear region exists in the cytoplasm, and the Ca clearance machinery is also likely clustered at selected locations. Therefore, during a prolonged depolarisation (lasting up to 1 s), Ca was able to reach the cell apical ciliated pole. The effective Ca diffusion constant, measured from the progression of Ca wavefronts in the cytosol, was about 57 micron2/s. Our results indicate that, in these hair cells, buffered diffusion of Ca 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.
