Binding of spermine and ifenprodil to a purified, soluble regulatory domain of the N-methyl-d-aspartate receptor
Binding of spermine and ifenprodil to a purified, soluble regulatory domain of the N-methyl-d-aspartate receptor
复制标题
DOI：
10.1111/j.1471-4159.2008.05729.x
复制
发表时间：
2008-12-01
影响因子：
4.7
通讯作者：
Igarashi, Kazuei
中科院分区：
文献类型：
--
作者：
Han, Xia;Tomitori, Hideyuki;Igarashi, Kazuei
用纯化的N-甲基-D-天冬氨酸受体NR1、NR2A和NR2B亚基的调节域NR1-R、NR2A-R和NR2B-R研究了精胺和异丙苯地尔与N-甲基-D-天冬氨酸受体氨基末端调节域的结合。R结构域在大肠杆菌中高效表达，纯化至接近均一的水平。[C-14]精胺与NR1-R、NR2A-R和NR2B-R结合的K-d值分别为19、140和33mU M。[H-3]在所测试的浓度(0.1-0.8 mU M)下，异芬普地尔与NR1-R(K-d，0.18 mU M)和NR2B-R(K-d，0.21 mU)结合，但不与NR2A-R结合。这些K-d值被圆二向色性测量所证实。K-d值反映了它们在完整的NR1/NR2A和NR1/NR2B受体上的有效浓度。结果提示，精胺和异丙苯地尔对NMDA受体的作用是通过与NR1、NR2A和NR2B亚单位的调节域结合而实现的。精胺或异丙苯地尔与NR1-R和NR2a-R或NR1-R和NR2B-R的结合能力与每个R结构域的结合能力是相加的。精胺与NR1-R和NR2B-R的结合不受异丙苯地尔的抑制，反之亦然，说明精胺和异丙苯地尔在NR1-R和NR2B-R上的结合部位不同。
The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N-methyl-D-aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1-R, NR2A-R and NR2B-R. The R domains were over-expressed in Escherichia coli and purified to near homogeneity. The K-d values for binding of [C-14]spermine to NR1-R, NR2A-R and NR2B-R were 19, 140, and 33 mu M, respectively. [H-3]Ifenprodil bound to NR1-R (K-d, 0.18 mu M) and NR2B-R (K-d, 0.21 mu M), but not to NR2A-R at the concentrations tested (0.1-0.8 mu M). These K-d values were confirmed by circular dichroism measurements. The K-d values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1-R and NR2A-R or NR1-R and NR2B-R was additive with that of each individual R domain. Binding of spermine to NR1-R and NR2B-R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1-R and NR2B-R are distinct.