A point mutation in the gamma(2) subunit of gamma-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity
A point mutation in the gamma(2) subunit of gamma-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity
复制标题
DOI：
10.1073/pnas.94.16.8824
复制
发表时间：
1997-08-05
影响因子：
11.1
通讯作者：
Sigel, E
中科院分区：
文献类型：
--
作者：
Buhr, A;Sigel, E
苯二氮卓类药物变构调节γ-氨基丁酸（GABA）诱发的γ-氨基丁酸A型（GABA（A））受体的氯电流。大鼠γ（2）或γ（3）与α（1）和β（2）亚基的共表达导致两种受体均显示高[H-3]Ro 15-1788亲和力。与含有γ（2）的受体相比，含有γ 3亚基的受体对唑吡坦的亲和力降低178倍。在γ（2）和γ（3）之间构建八个嵌合体，随后在γ（2）中构建九个不同的点突变，每个点突变与在γ（3）中发现的同源氨基酸残基。在人胚肾293细胞中，嵌合或突变的γ亚基与α（1）和β（2）共表达，以定位导致唑吡坦亲和力降低的氨基酸残基，γ（2）M130 L的130位的甲硫氨酸取代为亮氨酸导致唑吡坦亲和力降低51倍，而对[H-3]Ro 15-的亲和力降低51倍。1788保持不变，对地西泮的亲和力仅降低约2倍，saute突变导致Cl 213572亲和力增加9倍，发现第二个突变（γ（2）M57 I）使唑吡坦亲和力降低约4倍。野生型和γ（2）M130 L-含有受体在爪蟾卵母细胞中功能性表达。突变后，激动剂和调节位点之间的变构偶联得以保留，唑吡坦和地西泮的剂量-反应曲线显示，I-唑吡坦而不是地西泮的表观亲和力急剧降低，表观GABA亲和力不受γ（2）M130 L突变的显著影响，所鉴定的氨基酸残基可能定义了GABA（A）受体的苯二氮卓类结合口袋的一部分。由于GABA（A）受体中的调节位点与GABA位点以及相关受体的所有激动剂位点同源，因此γ（2）M130可能指向对所有受体中的激动剂结合重要的同源区域，或者定义了一个不基于该原理的新区域。
Benzodiazepines allosterically modulate gamma-aminobutyric acid (GABA) evoked chloride currents of gamma-aminobutyric acid type A (GABA(A)) receptors, Coexpression of either rat gamma(2) or gamma(3), in combination with alpha(1) and beta(2) subunits, results both in receptors displaying high [H-3]Ro 15-1788 affinity, However, receptors containing a gamma 3 subunit display a 178-fold reduced affinity to zolpidem as compared with gamma(2)-containing receptors. Eight chimeras between gamma(2) and gamma(3) were constructed followed by nine different point mutations in gamma(2), each to the homologous amino acid residue found in gamma(3). Chimeric or mutant gamma subunits were coexpressed with alpha(1) and beta(2) in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity, Substitution of a methionine-to-leucine at position 130 of gamma(2) (gamma(2)M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [H-3]Ro 15-1788 remained unchanged, The affinity for diazepam was only decreased by about 2-fold, The saute mutation resulted in a 9-fold increase in Cl 213572 affinity, A second mutation (gamma(2)M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and gamma(2)M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved, Dose-response curves for zolpidem and for diazepam showed that I-he zolpidem but not the diazepam apparent affinity is drastically reduced, The apparent GABA affinity is not significantly affected by the gamma(2)M130L mutation, The identified amino acid residues may define part of the benzodiazepine binding pocket of GABA(A) receptors. As the modulatory site in the GABA(A) receptor is homologous to the GABA site, and to all agonist sites of related receptors, gamma(2)M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.