CALCIUM CHANNEL MODULATION IN VESTIBULAR HAIR CELLS

The Ca2+ inflow through basolateral voltage-gated Ca2+ channels of cochlear and 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. It has been found that in many preparations the Ca2+ channels are regulated via a G protein or via a phosphorylation. Moreover, it has been suggested that prolonged depolarizations (>1sec) produce a long term inactivation of Ca2+ current in turtle vestibular hair cells, as found in other preparations. In the present work, the regulation of Ca2+ channels of semicircular canal vestibular hair cells was investigated. The experiments were performed on hair cells mechanically or enzymatically dissociated, recorded in the whole-cell configuration under visual control, using intra- and extracellular solutions designed to block all but the voltage-activated Ca2+ currents. In the presence of 1 mM ATP in the pipette solution, about 60% of the recorded cells displayed a Ca2+ current formed by an L-type and a drug-resistant (R2) components, while the remaining 40% also exhibited an additional drug-resistant fraction (R1), which inactivated in a Ca-dependent manner. If ATP was raised to 10 mM, the R1 component was progressively enhanced as intracellular ATP equilibrated with the pipette solution, and became apparent in all recordings. Nifedipine (5 mM) reduced the plateau amplitude by the same amount (70%), irrespective of the size attained following the run-up. This indicates that the run-up was targeted to the R1 and L current fractions, without affecting the R2 channel. In cells having the R1 component, high ATP increased the R1 and the L component of about 380% and about 170%, respectively. Cells initially lacking the R1 component had a similar increase in the L component, while the R1 component raised from 0 to about 25 pA. The addition of cGMP or cAMP (1 mM) to the internai solution, containing 1 or 10 mM ATP, did not cause any further increase of the current. It is concluded that intracellular ATP boosts the R1- and L-type current directly or via a cyclic-nucleotide independent phosphorylation. Despite the presence of intracellular ATP, long depolarizations (>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 internai Cs+ was substituted with NMG+, demonstrating that the decay was actually generated by an outward current carried by Cs+, possibly flowing through the Ca2+ channels. However, Ca2+ channel blockade probed during the current decay with the fast application of 200 microM Cd2+, reduced the total current by about the same amount. This shows that the decay was not produced by the progressive increase of an outward flow of Cs+ through the Ca2+ channels, reducing (or even cancelling) the Ca2+ inflow. Rather, the decay of the total current was instead generated by the progressive activation of an outward current flowing through another channel type. In conclusion, prolonged depolarizations do not cause the full inactivation of the Ca2+ current, but the decay of total current is mostly produced by the unblock of cationic channels (possibly selective to K+), carrying an outward Cs+ current, despite the presence of 20 mM TEA in all solutions.