Functional evidence of distinct ATP activation sites at the human P2X7 receptor
Functional evidence of distinct ATP activation sites at the human P2X7 receptor
复制标题
DOI：
10.1111/j.1469-7793.2001.00025.x
复制
发表时间：
2001-07-01
影响因子：
5.5
通讯作者：
Markwardt, F
中科院分区：
文献类型：
--
作者：
Klapperstück, M;Büttner, C;Markwardt, F
1.使用双微电极电压钳技术研究激动剂ATP对表达野生型人P2 X（7）受体（hP 2X（7））、N端六组氨酰标记的hP 2X（7）受体（His-hP 2X（7））或C端缺失156个氨基酸的截短型His-hP 2X（7）受体（His-hP 2X（7）DeltaC）的爪蟾卵母细胞全细胞电流的影响.野生型hP 2X（7）受体的激活时间过程可以描述为指数增长和额外的几乎线性激活电流分量的总和。野生型hP 2X（7）受体的指数激活电流分量的幅度显示出对激动剂浓度的双相依赖性，这可以通过两个相等的高灵敏度和两个相等的低灵敏度非合作激活位点的模型来最好地近似，其表观解离常数分别为约4和200 μ M游离ATP（4-）。线性激活电流单相依赖于激动剂浓度，表观解离常数约为200 μ M。在表达His-hP 2X（7）或His-hP 2X（7）DeltaC. 6的卵母细胞中，低敏感性位点对电流动力学的贡献减少或几乎消失。我们的数据表明，hP 2X（7）受体具有至少两种类型的激活位点，其在ATP 4敏感性上相差50倍。这两个位点的占据程度影响活化和失活动力学。N-和C-末端结构域似乎是由低ATP敏感性位点激活引起的电流的重要决定因素，但不是由高ATP敏感性位点介导的电流。
1. The effect of the agonist ATP on whole cell currents of Xenopus oocytes expressing: either the wild-type human P2X(7) receptor (hP2X(7)), an N-terminally hexahistidyl-tagged hP2X(7) receptor (His-hP2X(7)), or a truncated His-hP2X(7) receptor (His-hP2X(7)DeltaC) lacking the C-terminal 156 amino acids was investigated using the two-microelectrode voltage clamp technique.2. The activation time course of the wild-type hP2X(7), receptor can be described as the sum of an exponentially growing and an additional almost linearly activating current component.3. The amplitude of the exponentially activating current component of the wild-type hP2X(7) receptor displayed a biphasic dependence on the agonist concentration, which could be best approximated by a model of two equal high-sensitivity and two equal low-sensitivity noncooperative activation sites with apparent dissociation constants of about 4 and 200 muM free ATP(4-), respectively.4. The linearly activating current was monophasically dependent on the agonist concentration with an apparent dissociation constant of about 200 muM.5. The contribution of the low-sensitivity sites to current kinetics was reduced or almost abolished in oocytes expressing His-hP2X(7) or His-hP2X(7)DeltaC.6. Our data indicate that the hP2X(7) receptor possesses at least two types of activation sites, which differ in ATP4- sensitivity by a factor of 50. The degree of occupation of these two sites influences both activation and deactivation kinetics. Both N- and C-terminal domains appear to be important determinants of the current elicited by activation of the sites with low ATP sensitivity, but not for that mediated by the highly ATP-sensitive sites.