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+ cha nnels (Ca(V)2 channels) were investigated in cell-attached patches of nerve growth factor (NGF)-differentiated PC12 cells. Ca(V)2 channels, mainly com posed 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 muM B ayK8644, or L-type channel block by 5 muM nifedipine. At a test potential o f +20mV, they had a unitary current amplitude of similar to0.5 pA and open time constants of similar to0.4 ms and similar to1.1 ms when fit assuming d ouble exponential components. As bath application of amlodipine was ineffec tive to modify Ca(V)2 channels in the sealed patch, we analyzed the channel activity when giga-seal formation was obtained in the presence of amlodipi ne (10 muM both in the pipette and bath solution). Amlodipine did not modif y the unitary current amplitude but suppressed the channel open probability (NPo) when holding potential was depolarized, shifting the voltage-depende nt inactivation curve towards negative potentials by 25mV. Amlodipine-induc ed 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 amlodipin e, prevented channel openings through the high-affinity binding to the inac tivated state, as often observed when dihydropyridines block L-type Ca2+ ch annels.