Voltage-dependent modulation of single N-Type Ca2+ channel kinetics by receptor agonists in IMR32 cells

The voltage-dependent inhibition of single N-type Ca(2+) channels by noradrenaline (NA) and the delta-opioid agonist D-Pen(2)-D-Pen (5)-enkephalin (DPDPE) was investigated in cell-attached patches of human neuroblastoma IMR32 cells with 100 mM Ba(2+) and 5 microM nifedipine to block L-type channels. In 70% of patches, addition of 20 microM NA + 1 microM DPDPE delayed markedly the first channel openings, causing a four- to fivefold increase of the first latency at +20 mV. The two agonists or NA alone decreased also by 35% the open probability (P(o)), prolonged partially the mean closed time, and increased the number of null sweeps. In contrast, NA + DPDPE had little action on the single-channel conductance (19 versus 19.2 pS) and minor effects on the mean open time. Similarly to macroscopic Ba(2+) currents, the ensemble currents were fast activating at control but slowly activating and depressed with the two agonists. Inhibition of single N-type channels was effectively removed (facilitated) by short and large depolarizations. Facilitatory pre-pulses increased P(o) significantly and decreased fourfold the first latency. Ensemble currents were small and slowly activating before pre-pulses and became threefold larger and fast decaying after facilitation. Our data suggest that slowdown of Ca(2+) channel activation by transmitters is mostly due to delayed transitions from a modified to a normal (facilitated) gating mode. This single-channel gating modulation could be well simulated by a Monte Carlo method using previously proposed kinetic models predicting marked prolongation of first channel openings.