Kinetic Study of N-Type Calcium Current Modulation by δ-Opioid Receptor Activation in the Mammalian Cell Line NG108-15

Abstract

The voltage-dependent inhibition of N-type Ca 2+ channel current by the δ-opioid agonist [ d-pen 2, d-pen 5]-enkephalin (DPDPE) was investigated in the mammalian cell line NG108-15 with 10 μM nifedipine to block L-type channels, with whole-cell voltage clamp methods. In in vitro differentiated NG108-15 cells DPDPE reversibly decreased ω-conotoxin GVIA-sensitive Ba 2+ currents in a concentration-dependent way. Inhibition was maximal with 1 μM DPDPE (66% at 0 mV) and was characterized by a slowing of Ba 2+ current activation at low test potentials. Both inhibition and kinetic slowing were attenuated at more positive potentials and could be relieved up to 90% by strong conditioning depolarizations. The kinetics of removal of inhibition (de-inhibition) and of its retrieval (re-inhibition) were also voltage dependent. Both de-inhibition and re-inhibition were single exponentials and, in the voltage range from −20 to +10 mV, had significantly different time constants at a given membrane potential, the time course of re-inhibition being faster than that of de-inhibition. The kinetics of de-inhibition at −20 mV and of reinhibition at −40 mV were also concentration dependent, both processes becoming slower at lower agonist concentrations. The rate of de-inhibition at +80/+120 mV was similar to that of Ca 2+ channel activation at the same potentials measured during application of DPDPE (∼7 ms), both processes being much slower than channel activation in controls (<1 ms). Moreover, the amplitude but not the time course of tail currents changed as the depolarization to +80/+120 mV was made longer. The state-dependent properties of DPDPE Ca 2+ channel inhibition could be simulated by a model that assumes that inhibition by DPDPE results from voltage- and concentration-dependent binding of an inhibitory molecule to the N-type channel.