Effects of amlodipine on unitary non-L-type high voltage-activated Ca2+ channel currents in differentiated PC12 cells

Effects of amlodipine on unitary non-L-type high voltage-activated Ca2+ channels (Cav2 channels) were investigated in cell-attached patches of nerve growth factor (NGF)-differentiated PC12 cells. Cav2 channels, mainly composed of N-type channel in our experimental condition, were defined in this study as high voltage-activated Ca2+ channels obtained after selection of patches without L-type channel "mode 2" activity in the presence of 1 microM BayK8644, or L-type channel block by 5 microM nifedipine. At a test potential of +20 mV, they had a unitary current amplitude of approximately 0.5 pA and open time constants of approximately 0.4 ms and approximately 1.1 ms when fit assuming double exponential components. As bath application of amlodipine was ineffective to modify Cav2 channels in the sealed patch, we analyzed the channel activity when giga-seal formation was obtained in the presence of amlodipine (10 microM both in the pipette and bath solution). Amlodipine did not modify the unitary current amplitude but suppressed the channel open probability (NPo) when holding potential was depolarized, shifting the voltage-dependent inactivation curve towards negative potentials by 25mV. Amlodipine-induced suppression of NPo was mainly due to the decreased ratio of sweeps with channel openings (availability) and was not associated with changes in open time constants. These results were consistent with the view that amlodipine prevented channel openings through the high-affinity binding to the inactivated state, as often observed when dihydropyridines block L-type Ca2+ channels.