Han S H, Cho Y W, Kim C J, Min B I, Rhee J S, Akaike N
Department of Physiology, Kyunghee University College of Medicine, Seoul, South Korea.
Neuroscience. 1999 Apr;90(1):209-19. doi: 10.1016/s0306-4522(98)00409-6.
The characteristics of the inwardly rectifying K+ current activated by a mu-type opioid agonist, D-Ala2,N-MePhe4,Gly5-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 (GDPbetas, 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 (GTPgammas, 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 microM for 2 min, I(DAMGO) was completely abolished. When a conventional whole-cell patch was made with a patch-pipette containing 1 microg/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-l-methyl-xanthine and dibutyryladenosine 3'5'-cyclic monophosphate) and protein kinase C activators (phorbol-12-myristate-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 Gi and/or Go; and (iii) the G-proteins exert their roles in I(DAMGO) without any mediation of the second messenger systems.
采用制霉菌素穿孔膜片钳和传统全细胞膜片钳记录模式，在电压钳条件下，研究了μ型阿片类激动剂D - Ala2，N - MePhe4，Gly5 - ol - 脑啡肽（DAMGO）激活的内向整流钾电流在急性分离的大鼠导水管周围灰质神经元中的特性。DAMGO以浓度和电压依赖性方式激活内向电流。DAMGO诱导的电流是一种内向整流钾电流（I(DAMGO)），它对钾通道阻滞剂奎宁和Ba2 +敏感，但对Cs +和四乙铵不敏感。在传统全细胞膜片钳模式下，5'-O-(2 - 硫代二磷酸)三锂盐鸟苷（GDPβs，0.4 mM）将I(DAMGO)的幅度抑制至初始电流幅度的28%。在用5'-O-(3 - 硫代三磷酸)四锂盐鸟苷（GTPγs，0.4 mM）进行细胞内灌注1分钟后，首次应用DAMGO不可逆地激活了I(DAMGO)。通过细胞外应用浓度为50 μM的N - 乙基马来酰胺2分钟，I(DAMGO)被完全消除。当用含有1 μg/ml百日咳毒素和1 mMβ - 烟酰胺腺嘌呤二核苷酸的膜片电极进行传统全细胞膜片钳记录时，I(DAMGO)逐渐下降至其初始幅度的约41%。细胞外应用包括蛋白激酶抑制剂（星形孢菌素）、蛋白激酶A激活剂（福斯高林、3 - 异丁基 - 1 - 甲基 - 黄嘌呤和二丁酰腺苷3',5'-环一磷酸）和蛋白激酶C激活剂（佛波醇 - 12 - 肉豆蔻酸 - 13 - 乙酸酯和1 - 油酰基 - 2 - 乙酰 - sn - 甘油）的第二信使调节剂对I(DAMGO)没有影响。这些结果表明：（i）DAMGO激活的内向整流钾电流由百日咳毒素敏感的鸟嘌呤核苷酸结合蛋白（G蛋白）介导；（ii）参与I(DAMGO)的G蛋白类型为Gi和/或Go；（iii）G蛋白在I(DAMGO)中发挥作用，无需第二信使系统的任何介导。
