McKernan R M, Biggs C S, Gillard N, Quirk K, Ragan C I
Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, U.K.
Biochem J. 1990 Aug 1;269(3):623-8. doi: 10.1042/bj2690623.
The 5-HT3 hydroxytryptamine receptor from NCB 20 cells was solubilized and the molecular and hydrodynamic properties of the receptor were investigated. The receptor was identified by binding of the radioligand 3-NN'-[3H]dimethyl-8-azabicyclo[3.2.1]octanyl indol-3-yl carboxylate ester [( 3H]Q ICS 205-930) to NCB 20 membranes (Bmax = 1.19 +/- 0.31 pmol/mg of protein; Kd = 0.43 +/- 0.076 nM) and was optimally solubilized with 0.5% deoxycholate. [3H]Q ICS 205-930 labelled one population of sites in solution (Bmax = 1.11 +/- 0.4 pmol/mg of protein; Kd = 0.48 +/- 0.06 nM; n = 4). The characteristics of [3H]Q ICS 205-930 binding were essentially unchanged by solubilization, and competition for [3H]Q ICS 205-930 binding by a series of 5-HT3 agonists and antagonists was consistent with binding to a 5-HT3 receptor site and was similar to that observed for 5-HT3 receptors solubilized from rat brain [McKernan, Quirk, Jackson & Ragan (1990) J. Neurochem. 54, 924-930]. Some physical properties of the solubilized receptor were investigated. The molecular size (Stokes radius) of the [3H]Q ICS 205-930-binding site was measured by gel-exclusion chromatography in a buffer containing 0.2% Lubrol and 0.5 M-NaCl and was determined as 4.81 +/- 0.15 nm (mean +/- S.E.M.; n = 6). Sucrose-density-gradient centrifugation was also performed under the same detergent and salt conditions to determine the partial specific volume (v) of the detergent-receptor site complex. This was found to be 0.794 ml.g-1. Sucrose-density-gradient centrifugation was carried out in both 1H2O and 2H2O to allow correction for detergent binding to the receptor. The Mr of the 5-HT3 receptor under these conditions was calculated as 249,000 +/- 18,000 (n = 3). The size and physical properties of the 5-HT3 receptor are similar to those observed for members of the family of ligand-gated ion channels.
从NCB 20细胞中溶解5-羟色胺3（5-HT3）受体，并对该受体的分子和流体动力学性质进行了研究。通过放射性配体3-NN'-[3H]二甲基-8-氮杂双环[3.2.1]辛基吲哚-3-基羧酸酯[（3H]Q ICS 205-930）与NCB 20细胞膜的结合来鉴定该受体（Bmax = 1.19±0.31 pmol/mg蛋白质；Kd = 0.43±0.076 nM），并用0.5%脱氧胆酸盐将其最佳溶解。[3H]Q ICS 205-930在溶液中标记了一组位点（Bmax = 1.11±0.4 pmol/mg蛋白质；Kd = 0.48±0.06 nM；n = 4）。溶解后，[3H]Q ICS 205-930结合的特性基本不变，一系列5-HT3激动剂和拮抗剂对[3H]Q ICS 205-930结合的竞争与与5-HT3受体位点的结合一致，并且与从大鼠脑溶解的5-HT3受体观察到的情况相似[麦肯南、夸克、杰克逊和拉根（1990年）《神经化学杂志》54，924 - 930]。研究了溶解受体的一些物理性质。在含有0.2%十二烷基醇聚氧乙烯醚和0.5 M氯化钠的缓冲液中，通过凝胶排阻色谱法测量[3H]Q ICS 205-930结合位点的分子大小（斯托克斯半径），确定为4.81±0.15 nm（平均值±标准误；n = 6）。在相同的去污剂和盐条件下也进行了蔗糖密度梯度离心，以确定去污剂-受体位点复合物的偏比容（v）。发现其为0.794 ml·g-1。在1H2O和2H2O中都进行了蔗糖密度梯度离心，以便校正去污剂与受体的结合。在这些条件下，5-HT3受体的Mr计算为249,000±18,000（n = 3）。5-HT3受体的大小和物理性质与配体门控离子通道家族成员观察到的相似。
