Zhang J, Xiao Y, Abdrakhmanova G, Wang W, Cleemann L, Kellar K J, Morad M
Department of Pharmacology, Georgetown University School of Medicine, Washington, DC., USA.
Mol Pharmacol. 1999 Jun;55(6):970-81. doi: 10.1124/mol.55.6.970.
The alpha3/beta4 rat neuronal nicotinic acetylcholine receptor, stably transfected in human embryonic kidney cells, was examined using the whole-cell-clamp technique and 2-dimensional confocal imaging. Application of agonists (nicotine, cytisine, epibatidine) activated a large (100-200 pA/pF) inwardly rectifying monovalent current, with little current at voltages between 0 and +40 mV. Rapid application of nicotine and cytisine indicated EC50 values of congruent with22 and congruent with64 microM, respectively, and suggested second order binding kinetics (Hill coefficient approximately 2). The time constant of desensitization (decay) of nicotine-activated current was concentration-dependent (typically approximately 10 s at 30 microM versus approximately 1.0 s at 100-1000 microM), but not voltage-dependent and was significantly smaller than the approximately 200 s reported for the alpha3/beta4 receptor expressed in Xenopus oocytes. Nicotine-activated current was rapidly and reversibly blocked by coapplication of mecamylamine and d-tubocurarine. At -80 mV holding potentials, the current was also suppressed by approximately 25% either upon complete removal or elevation of Ca2+ to 10 mM. Total replacement of Na+ by Ca2+ also completely blocked the current. On the other hand, evidence for permeation of Ca2+ was indicated by increased inward current at -40 mV upon elevation of Ca2+ from 2 to 10 mM, as well as a rise in the cytosolic Ca2+ proportional to the current carried by the receptor. These findings are consistent with the idea that Ca2+, in addition to its channel-permeating properties, may also regulate the receptor from an extracellular site. Our results suggest that the alpha3/beta4 neuronal nicotinic acetylcholine receptor, when stably expressed in human embryonic kidney 293 cells, has desensitization kinetics and Ca2+ regulatory mechanisms somewhat different from those described for the receptor expressed in Xenopus oocytes.
利用全细胞膜片钳技术和二维共聚焦成像技术，对稳定转染于人胚肾细胞中的α3/β4大鼠神经元烟碱型乙酰胆碱受体进行了研究。应用激动剂（尼古丁、金雀花碱、埃博霉素）可激活一个大的（100 - 200 pA/pF）内向整流单价电流，在0至 +40 mV之间的电压下电流很小。快速应用尼古丁和金雀花碱表明，其半数有效浓度（EC50）值分别约为22 μM和约64 μM，并提示二级结合动力学（希尔系数约为2）。尼古丁激活电流的脱敏（衰减）时间常数呈浓度依赖性（在30 μM时通常约为10 s，而在100 - 1000 μM时约为1.0 s），但不依赖电压，且明显小于在非洲爪蟾卵母细胞中表达的α3/β4受体所报道的约200 s。尼古丁激活电流可被联合应用美加明和d - 筒箭毒碱快速且可逆地阻断。在 -80 mV的钳制电位下，完全去除Ca2+或将Ca2+升高至10 mM时，电流也会被抑制约25%。用Ca2+完全替代Na+也会完全阻断电流。另一方面，当Ca2+从2 mM升高至10 mM时，在 -40 mV处内向电流增加，以及胞质Ca2+的升高与受体携带的电流成比例，这表明存在Ca2+通透的证据。这些发现与以下观点一致，即Ca2+除了具有通道通透特性外，还可能从细胞外位点调节受体。我们的结果表明，当α3/β4神经元烟碱型乙酰胆碱受体在人胚肾293细胞中稳定表达时，其脱敏动力学和Ca2+调节机制与在非洲爪蟾卵母细胞中表达的该受体所描述的有所不同。
