μ-opioid agonist-induced activation of G-protein-coupled inwardly rectifying potassium current in rat periaqueductal gray neurons

The characteristics of the inwardly rectifying K+ current activated by a mu-type opioid agonist, D-Ala(2),N-MePhe(4),Gly(5)-ol-enkephalin (DAMGO), were examined in the acutely dissociated rat periaqueductal gray neurons using the nystatin-perforated and the conventional whole-cell recording modes under voltage-clamp conditions. DAMGO activated inward currents in a concentration- and voltage-dependent manner. The DAMGO-induced current was an inwardly rectifying K+ current (I-DAMGO) which was sensitive to K+ channel blockers, quinine and Ba2+ but insensitive to Cs+ and tetraethylammonium. In the conventional whole-cell clamp mode, guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDP beta s, 0.4 mM) inhibited the amplitude of I-DAMGO to 28% Of that of the initial current. After the intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate) tetralithium salt (GTP gamma s, 0.4 mM) for 1 min, the first application of DAMGO irreversibly activated I-DAMGO. By the extracellular application of N-ethylmaleimide at a concentration of 50 mu M for 2 min, I-DAMGO was completely abolished. When a conventional whole-cell patch was made with a patch-pipette containing 1 mu g/ml of pertussis toxin together with 1 mM of beta-nicotinamide adenine dinucleotide, I-DAMGO gradually declined to about 41% of its initial amplitude. The extracellular application of second messenger modulators including protein kinase inhibitor (staurosporin), protein kinase A activators (forskolin, 3-isobutyl-1-methyl-xanthine and dibutyryladenosine 3'5'-cyclic monophosphate) and protein kinase C activators (phorbol-12-myristale-13-acetate and 1-oleoyl-2-acetyl-sn-glycerol) had no effect on I-DAMGO. These results suggest that (i) DAMGO-activated inwardly rectifying K+ current is mediated by pertussis toxin-sensitive guanine nucleotide binding proteins (G-proteins); (ii) the types of G protein involved in I-DAMGO are G(i) and/or G(o); and (iii) the G-proteins exert their roles in I-DAMGO without any mediation of the second messenger systems. (C) 1999 IBRO. Published by Elsevier Science Ltd.