Photoaffinity Labeling of the GABAA Receptor with [3H]Muscimol
Photoaffinity Labeling of the GABAA Receptor with [3H]Muscimol
复制标题
用 [3H]Muscimol 光亲和标记 GABAA 受体
作者：
D. Cavalla;N. Neff
摘要： 蝇蕈醇是γ-氨基丁酸A (GABAA) 受体最有效的激动剂配体之一。对其化学结构的分析表明它是光亲和标记的候选者。在实践中，254 nm 的紫外线照射既改变了蝇蕈醇的紫外线光谱，又诱导[3H]-蝇蕈醇与大鼠小脑突触体膜不可逆结合。照射 10 分钟后，使用 10 nM [3H]muscimol，该结合的特定部分为 270 fmol/mg 蛋白质。 （非特异性结合定义为在 1 mM GABA 存在下产生的结合。）特异性结合逐渐增加至 100 nM [3H]muscimol。膜本身的辐射不会显着改变可逆[3H]蝇蕈醇结合的KD 或Bmax。然而，[3H]蝇蕈醇的照射使其随后对膜进行光标记的能力降低了86±3%。观察到蝇蕈醇、GABA 和荷包牡丹碱的结合剂量依赖性抑制； 10 nM [3H]muscimol 的最大抑制是总标记的 70%，这三种化合物的效力顺序是 GABAA 受体标记的特征。巴氯芬、L-谷氨酸和地西泮在高浓度下没有作用。光标记膜的 SDS-PAGE 表明放射性特异性掺入两种分子量物质中。其中一种未能进入分离凝胶，这意味着分子量 >250,000 道尔顿 (250 kD)。通过荧光照相法鉴定另一种的分子量约为 52,000 道尔顿 (52 kD)。
Abstract: Muscimol is one of the most potent agonist ligands at the γ‐aminobutyric acidA (GABAA) receptor. Analysis of its chemical structure showed it to be a candidate for photoaffinity labeling. In practice, UV irradiation at 254 nm both changed the UV spectrum of muscimol and induced an irreversible binding of [3H]‐muscimol to rat cerebellar synaptosomal membrane. After 10 min of irradiation, using 10 nM [3H]muscimol, the specific portion of this binding was 270 fmol/mg protein. (Nonspecific binding was defined as that arising in the presence of 1 mM GABA.) Specific binding increased asymptotically up to 100 nM [3H]muscimol. Irradiation of the membranes themselves did not significantly alter the KD or Bmax of reversible [3H]muscimol binding. However, irradiation of [3H]muscimol reduced its capacity subsequently to photolabel the membranes by 86 ± 3%. Dose‐dependent inhibition of binding was observed with muscimol, GABA, and bicuculline methiodide; with 10 nM [3H]muscimol maximum inhibition was 70% of total labeling and the order of potencies of these three compounds was characteristic of labeling to the GABAA receptor. Baclofen, l‐glutamate, and diazepam exerted no effect at high concentrations. SDS‐PAGE of the photolabeled membranes indicated specific incorporation of radioactivity into two molecular‐weight species. One failed to enter the separating gel, implying a molecular weight >250,000 daltons (250 kD). The molecular weight of the other was identified by fluorography to be about 52,000 daltons (52 kD).