Conductance properties of Ca2+ channels of vestibular hair cell are strongly affected by intracellular Ca2+ buffers

The effects of endogenous and exogenous Ca2+ buffers on the Ca2+ current kinetics were investigated in hair cells mechanically isolated from frog semicircular canals by using the perforated and whole-cell configurations of the patch clamp technique. As previously demonstrated in this preparation, in 4 mM external Ca2+ and in the presence of K+ channel blockers, patch recordings showed a Ca2+ current flowing through three different channel types: an initial transient current through a drug-resistant channel (R1), and a plateau fraction comprised of a large L component and a small drug-resistant current fraction (R2). In the perforated patch conditíon a large and stable Ca2+ current was recorded, with all three components. Cell dialysis with a buffer-free pipette solution did not prevent a complete Ca2+ response, although the size of the transient and plateau current fractions were greatly reduced. The addition of 5 mM EGTA to the pipette solution partially restored both current amplitude and waveform without modifying the ratio between the transient and the steady-state current fractions. In all these experimental conditions similar activation, inactivation and deactivation kinetics were observed. With 50 mM EGTA in the pipette solution, all the kinetic parameters were slowed down and the transient component increased in size with respect to that recorded in the absence of buffers or in the presence of 5 mM EGTA. No increase was detected in the plateau current. When 30 mM Cs-BAPTA was employed as a Ca2+ buffer, Ca2+ response kinetics were slowed down even more than in 50 mM EGTA experiments, and the Ca2+ waveform was substantially modified. A very large initial transient component appeared, which slowly but completely inactivated, leaving a plateau fraction of very small size, that deactivated along a single exponential time course with a slow time constant (suggesting that a single channel type - R2 - was still active at the end of the test). Under this condition the application of 10 microM nifedipine produced a great reduction of the transient current, leaving the plateau. component - and the deactivation phase - unaltered. The markedly reduced, remaining transient component presumably flowed through the R1 channels, and dísplayed a complete inactivation, slower than observed in the absence of high buffer levels. These results confirm that, at differente with cochlear hair cells, the frog semicircular canal receptors are endowed with several channel types, and highlight a dramatic alteration of L-type channel behavior and kinetic properties, when intracellular Ca2+ buffers are sufficiently concentrated and fast to interfere with rapid and local changes in Ca2+ levels.