Kusama T, Wang J B, Spivak C E, Uhl G R
Molecular Neurobiology Branch, National Institute on Drug Abuse, Baltimore, MD 21224.
Neuroreport. 1994 Jun 2;5(10):1209-12. doi: 10.1097/00001756-199406020-00012.
Seventeen site-directed mutations were constructed in the GABA rho 1 receptor with the aim of finding agonist binding domains common to rho 1 and rho 2 receptors but distinct from those identified in members of the family of homologous, ligand gated ion channels. Mutated cDNAs were expressed in Xenopus oocytes and tested by voltage clamp experiments. Five of the mutations abolished responsiveness to GABA. Mutation Q189H, in the conserved cysteine loop, diminished apparent GABA affinity to about 1/10 of wild type values in a manner consistent with decreased allosteric cooperativity among agonist recognition sites. Mutation R316A, located in the extracellular loop between transmembrane domains II and III, increased the Hill coefficient to 3.9 in a fashion consistent with enhanced open probability of a receptor multimer.
在GABA ρ1受体中构建了17个定点突变，目的是寻找ρ1和ρ2受体共有的激动剂结合结构域，但不同于在同源配体门控离子通道家族成员中鉴定出的结构域。突变的cDNA在非洲爪蟾卵母细胞中表达，并通过电压钳实验进行测试。其中五个突变消除了对GABA的反应性。保守半胱氨酸环中的Q189H突变使表观GABA亲和力降低至野生型值的约1/10，其方式与激动剂识别位点之间变构协同性降低一致。位于跨膜结构域II和III之间细胞外环中的R316A突变以与受体多聚体开放概率增加一致的方式将希尔系数提高到3.9。
