Calcium inflow through basolateral voltage-gated calcium channels of cochlear and vestibular hair cells sustains transmitter release at the cytoneural junction. It is therefore of key importance to establish how these channels are regulated by the voltage and/or by intracellular factors. To this aim, hair cells mechanically or enzymatically isolated from semicircular canals of the frog (Rana esculenta), were recorded in the whole-cell configuration under visual control, using intra- and extracellular solutions desígned to block all but the voltage-activated calcium currents. The extemal solution was changed rapidly (typically in less than 100 ms) by moving horizontally with a computer controlled stepping motor a multibarrelled perfusion pipette placed in front of the recorded celi. In the presence of 1 mM of ATP in the pipette solution, about 60% of the cells recorded in whole-cell mode displayed a calcium current formed by an L- and a drug-resistant (R2) components, while the remaining 40% also exhibited an additional drug-resistant fraction (R1), which inactivated in a calcium-dependent manner. If the pipette ATP was raised up to 10 mM, the R1 component was progressively enhanced as intracellular ATP equilibrate with the pipette solution, and became apparent in all recordings (instead of in 40% of them). In cells having the R1 component, the ATP dialysis produced an increase of R1 component of about 380%, whereas the L and the R2 component increased of about 170%. Cells initially lacking the R1 component had a similar increase in the R2 and L component, while the R1 component raised from 0 to about 25 pA. This indicate that ATP modulation was mainly targeted to the R1 channel. Despite the presence of intracellular ATP, very long depolarizations (larger than 5 s) produced a progressive decay of current to a steady state level: larger the depolarization, faster the decay. The steady level was usually outward for positive potentials (+20 mV); the decay was fully reversible upon returning to the holding potential. A steady inward current was recorded if the internal caesium was substituted with NMG, demonstrating that the decay was actually generated by an outward current carried by caesium, possibly flowing through the calcium channels. However, calcium channel blockade probed during the current decay with the fast application of 200 micromolar cadmium reduced the total current of the same amount. This shows that the decay was not produced by the progressive increase of an outward flow of caesium through the calcium channels, reducing (or even canceling) the calcium inflow. Rather, the decay of the total current was instead generated by the progressive activation of an outward current flowing through another channel type. One possibility is that long depolarizations unblock the potassium channels, that are therefore able to carry an outward caesium current.
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