Gong Yuan, Jiang Ji-Hong, Li Shi-Tong
Department of Anesthesiology, First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200030, P.R. China.
Mol Med Rep. 2016 Sep;14(3):2257-63. doi: 10.3892/mmr.2016.5493. Epub 2016 Jul 11.
The functional expression of recombinant α7 nicotinic acetylcholine receptors in human embryonic kidney (HEK) 293 cells has presented a challenge. Resistance to inhibitors of cholinesterase 3 (RIC‑3) has been confirmed to act as a molecular chaperone of nicotinic acetylcholine receptors. The primary objectives of the present study were to investigate whether the co‑expression of human (h)RIC‑3 with human α7 nicotinic acetylcholine receptor in HEK 293 cells facilitates functional expression of the α7 nicotinic acetylcholine receptor. Subsequent to transfection, western blotting and polymerase chain reaction were used to test the expression of α7 nicotinic acetylcholine receptor and RIC-3. The α7 nicotinic acetylcholine receptor was expressed alone or co‑expressed with hRIC‑3 in the HEK 293 cells. Drug‑containing solution was then applied to the cells via a gravity‑driven perfusion system. Calcium influx in the cells was analyzed using calcium imaging. Nicotine did not induce calcium influx in the HEK 293 cells expressing human α7 nicotinic acetylcholine receptor only. However, in the cells co‑expressing human RIC‑3 and α7 nicotinic acetylcholine receptor, nicotine induced calcium influx via the α7 nicotinic acetylcholine receptor in a concentration‑dependent manner (concentration required to elicit 50% of the maximal effect=29.21 µM). Taken together, the results of the present study suggested that the co‑expression of RIC‑3 in HEK 293 cells facilitated the functional expression of the α7 nicotinic acetylcholine receptor.
重组α7烟碱型乙酰胆碱受体在人胚肾（HEK）293细胞中的功能性表达一直是个挑战。胆碱酯酶3抑制剂抗性蛋白（RIC-3）已被证实可作为烟碱型乙酰胆碱受体的分子伴侣。本研究的主要目的是调查在HEK 293细胞中，人（h）RIC-3与人α7烟碱型乙酰胆碱受体共表达是否有助于α7烟碱型乙酰胆碱受体的功能性表达。转染后，采用蛋白质免疫印迹法和聚合酶链反应检测α7烟碱型乙酰胆碱受体和RIC-3的表达。α7烟碱型乙酰胆碱受体单独表达或与hRIC-3在HEK 293细胞中共表达。然后通过重力驱动灌注系统将含药溶液施加于细胞。使用钙成像分析细胞内的钙内流。尼古丁在仅表达人α7烟碱型乙酰胆碱受体的HEK 293细胞中未诱导钙内流。然而，在共表达人RIC-3和α7烟碱型乙酰胆碱受体的细胞中，尼古丁以浓度依赖性方式通过α7烟碱型乙酰胆碱受体诱导钙内流（产生最大效应50%所需的浓度=29.21µM）。综上所述，本研究结果表明，RIC-3在HEK 293细胞中的共表达促进了α7烟碱型乙酰胆碱受体的功能性表达。
