Kinetic study of N-type calcium current modulation by delta-opioid receptor activation in the mammalian cell line NG108-15.
The voltage-dependent inhibition of N-type Ca2+ channel current by the delta-opioid agonist [D-pen2, D-pen5]-enkephalin (DPDPE) was investigated in the mammalian cell line NG108-15 with 10 microM nifedipine to block L-type channels, with whole-cell voltage clamp methods. In in vitro differentiated NG108-15 cells DPDPE reversibly decreased omega-conotoxin GVIA-sensitive Ba2+ currents in a concentration-dependent way. Inhibition was maximal with 1 microM DPDPE (66% at 0 mV) and was characterized by a slowing of Ba2+ 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 Ca2+ channel activation at the same potentials measured during application of DPDPE (approximately 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 Ca2+ 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.
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Istituto di Fisiologia Generale, Universita' di Pavia and Istituto Nazionale per la Fisica della Materia, Pavia Unit, I-27100 Pavia, Italy. mtoselli@ipv36.unipv.it
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D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000145:classification / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D020881:Enkephalin, D-Penicillamine (2,5)-; D004745:Enkephalins / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials; D008954:Models, Biological; D009543:Nifedipine / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D017465:Receptors, Opioid, delta / Q000819:agonists / Q000378:metabolism*; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004745:Enkephalins; D010455:Peptides; D017465:Receptors, Opioid, delta; D020881:Enkephalin, D-Penicillamine (2,5)-; D020866:omega-Conotoxin GVIA; D009543:Nifedipine