Regulation Of κ-opioid Receptor Agonist U50488 On Potassium Channels In Undifferentiated PC12 Cells And Pilot Study Of Its Regulatory Mechanism

Outwardly rectifying potassium channels (Ik) are ubiquitously found in many mammalian cells, including undifferentiated clonal pheochromocytoma (PC12) cells. These channels, which play important roles in cellular signaling processes such as neurotransmitter release, membrane excitability, proliferation, and apoptosis, have been further explored based on recent knowledge of the structure of eukaryotic potassium channel.It is well known that several ligands of different G-protein coupled receptors (GPCRs) can alter the function of outward potassium channels. Stimulation of opioid receptors (μ, δ, and κ) that belong to a family of seven-transmembrane GPCRs leads to numerous effects through pertussis toxin (PTX)-sensitive G_i/o protein.This study was undertaken to determine the effect of U50488, a κ-opioid receptor agonist, on outwardly rectifying potassium channel (Ik) in undifferentiated PC12 cells.In the first series of experiments, the electrophysiological properties of Ik were characterized. Rectifying character of the voltage-dependent outward current is revealed. Outward currents measured at + 60 mV depolarizing voltage were markedly suppressed by adding 5 mM tetraethylammonium (TEA), and were resistant to 4-aminopridine (4-AP) at 5 mM concentration.In the second series of experiments, the effect of κ-opioid agonist (U50488) was investigated. Using whole-cell, on-cell and performed patch-clamp techniques, we found that U50488 decreased Ik amplitude in a time-dependent manner and Ik activation was delayed. Single-channel kinetics analysis provided a two-stage model for us to illuminate the blockage effect induced by U50488. The changes of kinetic properties of Ik were considered to be due to three types of close time: short close time, middle close time and long close time. At the first stage, the weight of short time constant (50.2 %, 1.07ms) increased to 76.2% (0.95ms), but the weights of middle (39.5%, 26.68ms) and long time constants (10.3%, 239ms) decreased to 21.6% (14.01ms) and 2.2% (249.5ms), respectively. The weight of long time constant...