Ca2+ inflow through voltage-gated Ca2+ channels of vestibular hair cells sustains transmitter release at the cytoneural junction. It is therefore important to establish how these channels are regulated by voltage and/or by intracellular factors. Hair cells isolated from frog semicircular canals were recorded in the whole-cell configuration, using intra- and extra-cellular solutions designed to block all but the voltage-gated Ca currents. In the presence of 1 mM ATP in the pipette, about 60 % of the cells displayed a Ca2+ current formed by a mix of L- and drug-resistant (R2) components, while the remaining 40 % exhibited an additional drug-resistant fraction (R1), which inactivated in a Ca2+-dependent manner. If the pipette ATP was raised up to 10 mM, R1 progressively enhanced as intracellular ATP equilibrated with the pipette solution, and became apparent in all recordings (instead of in 40 % of them). In cells exhibiting the R1 fraction, ATP dialysis produced a mean increase of the R1 component of about 380%., whereas L and R2 increase was of about 170 %. Cells initially lacking the R1 current had a similar raise in R2 and L fractions, while R1 raised from 0 to about 25 pA. During the ATP effect, cell perfusion with 10 microM nifedipine produced a 70 % decrease of the plateau current without affecting R1, as in control experiments. These resuits indicate that ATP modulation was mainly targeted to the R1 channel. Despite the presence of internal ATP, long depolarizations (>5 s) produced a decay of the current to a steady level: larger the depolarization, faster the decay. The steady level was outward at +20 mV; the decay was fully reversible on returning to the holding potential. Ca channel blockade, probed during current decay with the fast application of 200 microM Cd, reduced the total current of the same amount. This shows that the decay was not produced by the increase of outward Cs+ flow through the Ca2+ channel, reducing Ca2+ inflow. Rather, total current decay was generated by the progressive activation of an outward current flowing through a different channel type. Long depolarizations might unblock the K+ channels, which become able to carry outward Cs+ current.
Modulation of voltage-gated Ca2+ channels of vestibular hair cells
ROSSI, Marialisa;MARTINI, Marta;FARINELLI, Federica;RISPOLI, Giorgio
2005
Abstract
Ca2+ inflow through voltage-gated Ca2+ channels of vestibular hair cells sustains transmitter release at the cytoneural junction. It is therefore important to establish how these channels are regulated by voltage and/or by intracellular factors. Hair cells isolated from frog semicircular canals were recorded in the whole-cell configuration, using intra- and extra-cellular solutions designed to block all but the voltage-gated Ca currents. In the presence of 1 mM ATP in the pipette, about 60 % of the cells displayed a Ca2+ current formed by a mix of L- and drug-resistant (R2) components, while the remaining 40 % exhibited an additional drug-resistant fraction (R1), which inactivated in a Ca2+-dependent manner. If the pipette ATP was raised up to 10 mM, R1 progressively enhanced as intracellular ATP equilibrated with the pipette solution, and became apparent in all recordings (instead of in 40 % of them). In cells exhibiting the R1 fraction, ATP dialysis produced a mean increase of the R1 component of about 380%., whereas L and R2 increase was of about 170 %. Cells initially lacking the R1 current had a similar raise in R2 and L fractions, while R1 raised from 0 to about 25 pA. During the ATP effect, cell perfusion with 10 microM nifedipine produced a 70 % decrease of the plateau current without affecting R1, as in control experiments. These resuits indicate that ATP modulation was mainly targeted to the R1 channel. Despite the presence of internal ATP, long depolarizations (>5 s) produced a decay of the current to a steady level: larger the depolarization, faster the decay. The steady level was outward at +20 mV; the decay was fully reversible on returning to the holding potential. Ca channel blockade, probed during current decay with the fast application of 200 microM Cd, reduced the total current of the same amount. This shows that the decay was not produced by the increase of outward Cs+ flow through the Ca2+ channel, reducing Ca2+ inflow. Rather, total current decay was generated by the progressive activation of an outward current flowing through a different channel type. Long depolarizations might unblock the K+ channels, which become able to carry outward Cs+ current.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
