Intracellular Ca2+ buffers can dramatically affect Ca2+ conductances in hair cells

The effects of endogenous and exogenous Ca(2+) buffers on Ca(2+) current kinetics have been investigated by patch clamp in hair cells mechanically isolated from frog semicircular canals. This preparation displays at least three different Ca(2+) channel types: transient currents flow through a drug-resistant channel ("R1"), while non-inactivating channels sustain a steady, plateau current comprised of a large L component and a small drug-resistant fraction ("R2"). In the perforated-patch condition a large and stable Ca(2+) current was recorded, with all three components. In whole-cell, a buffer-free pipette solution did not prevent a complete Ca(2+) response. The size of the transient and plateau current fractions were greatly reduced, but the ratio between the two fractions, as well as the activation, inactivation and deactivation kinetics, were substantially unmodified. Current amplitude partially recovered with 5 mM EGTA in the pipette solution. With 50 mM EGTA all the kinetic parameters were slowed down and the transient component, but not the plateau component, markedly increased in size. Response kinetics slowed down even more with 30 mM Cs-BAPTA and the Ca(2+) waveform was substantially modified. The transient component was very large and inactivated slowly; the remaining very small plateau fraction deactivated along a slow, single exponential time. Under this condition nifedipine (10 microM) produced a great reduction of the transient current, leaving plateau and deactivation phase unaltered. This suggests that only R2 channels were still active at the end of the test and that the minor remaining transient component flowed through slowly but completely inactivating R1 channels. These results confirm the presence of several channel types in semicircular canal receptors, at difference with cochlear hair cells, and highlight a dramatic alteration of L-type channel behavior when intracellular Ca(2+) buffers are sufficiently concentrated and fast to interfere with rapid and local changes in Ca(2+) levels.