Schrattenholz A, Pfeiffer S, Pejovic V, Rudolph R, Godovac-Zimmermann J, Maelicke A
Laboratory of Molecular Neurobiology, Institute of Physiological Chemistry and Pathobiochemistry, 6 Duesbergweg, Johannes-Gutenberg University Medical School, 55099 Mainz, Germany.
J Biol Chem. 1998 Dec 4;273(49):32393-9. doi: 10.1074/jbc.273.49.32393.
The N-terminal extracellular region (amino acids 1-209) of the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% alpha-helical and 45% beta-structure. The fragment bound alpha-[3H]bungarotoxin in 1:1 stoichiometry with a KD value of 0.5 nM as determined from kinetic measurements (4 nM from equilibrium binding). The kinetics of association of toxin and fragment were of second order, with a similar rate constant (8.2 x 10(5) M-1 s-1) as observed previously for the membrane-bound heteropentameric nAChR. Binding of small ligands was demonstrated by competition with alpha-[3H]bungarotoxin yielding the following KI values: acetylcholine, 69 microM; nicotine, 0.42 microM; anatoxin-a, 3 miroM; tubocurarine, 400 microM; and methyllycaconitine, 0.12 microM. The results demonstrate that the N-terminal extracellular region of the nAChR alpha-subunit forms a self-assembling domain that functionally expresses major elements of the ligand binding sites of the receptor.
利用pET 3a载体，将来自电鳐电组织的烟碱型乙酰胆碱受体（nAChR）α亚基的N端细胞外区域（氨基酸1 - 209）在大肠杆菌中表达为包涵体。采用一种新的展开和重折叠方案，在无去污剂的情况下，获得了一种25 kDa的水溶性球状蛋白，其显示约15%的α螺旋结构和45%的β结构。根据动力学测量确定，该片段与α-[3H]银环蛇毒素以1:1化学计量比结合，KD值为0.5 nM（平衡结合时为4 nM）。毒素与片段结合的动力学为二级反应，其速率常数（8.2×10⁵ M⁻¹ s⁻¹）与先前观察到的膜结合异源五聚体nAChR相似。通过与α-[3H]银环蛇毒素竞争证明了小配体的结合，得到以下KI值：乙酰胆碱，69 μM；尼古丁，0.42 μM；anatoxin-a，3 μM；筒箭毒碱，400 μM；甲基lycaconitine，0.12 μM。结果表明，nAChRα亚基的N端细胞外区域形成了一个自组装结构域，该结构域在功能上表达了受体配体结合位点的主要元件。
