Liu L X, Burgess L H, Gonzalez A M, Sibley D R, Chiodo L A
Department of Pharmacology, Texas Tech University Health Science Center, Lubbock 79430, USA.
Synapse. 1999 Feb;31(2):108-18. doi: 10.1002/(SICI)1098-2396(199902)31:2<108::AID-SYN3>3.0.CO;2-V.
We utilized the approach of stably expressing different dopamine (DA) receptors into identified cell lines in an attempt to better understand the coupling of these receptors to membrane ion channels via second messenger systems. Recently, we examined the N18TG2 x mesencephalon (MES-23.5) cell line that is phenotypically similar to mesencephalic dopamine-containing neurons. Whole-cell voltage-clamp methods were used to investigate a voltage-dependent K+ current present in these cells. Untransfected MES-23.5 cells displayed a voltage-dependent slow-onset, slowly inactivating outward current which was not altered by bath application of either the D2 DA receptor agonist quinpirole (QUIN; 10-100 microM) or the D1 DA receptor agonist SKF38393, indicating that these cells were devoid of DA receptors. The K+ current studied was activated upon depolarization from a holding potential of -60 mV to a level more positive than -20 mV and was observed to be sensitive to bath application of tetraethylammonium. When MES-23.5 cells were transfected to stably express the D2S, D2L, D3, and D4 receptors, the same current was observed. In cells expressing D2L, D2S, and D3 receptors, application of the DA receptor agonists QUIN (1-80 microM), 7-hydroxy-dipropylaminoteralin (7-OH-DPAT, 1-80 microM), and dopamine (DA, 1-80 microM), increased the peak outward current by 35-40%. In marked contrast, cells stably expressing the D4 receptor demonstrated a significant DA agonist-induced reduction of the peak K+ current by 40%. For all four receptor subtypes, the D2-like receptor antagonist sulpiride (SUL 5 microM), when coapplied with QUIN (10 microM), totally abolished the change in K+ current normally observed, while coapplication of the D1-like receptor antagonist SCH23390 was without effect. The modulation of K+ current by D2L, D3, and D4 receptor stimulation was prevented by pretreatment of the cells with pertussis toxin (PTX, 500 ng/ml for 4 h). In addition, the intracellular application of a polyclonal antibody which specifically recognizes Goalpha completely blocked the ability of D2L, D3, and D4 receptors to modulate outward K+ currents. In contrast, the intracellular application of an antibody directed against Goalpha was without effect, whereas intracellular application of an antibody recognizing Gsalpha abolished the ability of the D2S receptor to enhance K+ current. These findings demonstrate that different members of the D2 DA receptor family may couple in a given cell to a common effector in dramatically different ways.
我们采用将不同多巴胺（DA）受体稳定表达于特定细胞系的方法，试图更好地理解这些受体通过第二信使系统与膜离子通道的偶联。最近，我们研究了N18TG2 x中脑（MES - 23.5）细胞系，其表型与中脑含多巴胺神经元相似。采用全细胞电压钳方法研究这些细胞中存在的电压依赖性钾电流。未转染的MES - 23.5细胞表现出电压依赖性的缓慢起始、缓慢失活的外向电流，浴加D2 DA受体激动剂喹吡罗（QUIN；10 - 100 microM）或D1 DA受体激动剂SKF38393对此电流均无影响，表明这些细胞缺乏DA受体。所研究的钾电流在从 - 60 mV的钳制电位去极化到比 - 20 mV更正的值时被激活，并且观察到其对浴加四乙铵敏感。当MES - 23.5细胞被转染以稳定表达D2S、D2L、D3和D4受体时，观察到相同的电流。在表达D2L、D2S和D3受体的细胞中，施加DA受体激动剂QUIN（1 - 80 microM）、7 - 羟基 - 二丙基氨基四氢萘（7 - OH - DPAT，1 - 80 microM）和多巴胺（DA，1 - 80 microM），可使外向电流峰值增加35 - 40%。与之形成显著对比的是，稳定表达D4受体的细胞表现出DA激动剂诱导的钾电流峰值显著降低40%。对于所有四种受体亚型，D2样受体拮抗剂舒必利（SUL 5 microM）与QUIN（10 microM）共同施加时，完全消除了通常观察到的钾电流变化，而共同施加D1样受体拮抗剂SCH23390则无作用。用百日咳毒素（PTX，500 ng/ml处理4小时）预处理细胞可阻止D2L、D3和D4受体刺激对钾电流的调节。此外，细胞内施加特异性识别Goα的多克隆抗体完全阻断了D2L、D3和D4受体调节外向钾电流的能力。相比之下，细胞内施加针对Goα的抗体则无作用，而细胞内施加识别Gsα的抗体则消除了DSS受体增强钾电流的能力。这些发现表明，D2 DA受体家族的不同成员在给定细胞中可能以截然不同的方式与共同效应器偶联。
