Xu W, Chen C, Huang P, Li J, de Riel J K, Javitch J A, Liu-Chen L Y
Department of Pharmacology and Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Biochemistry. 2000 Nov 14;39(45):13904-15. doi: 10.1021/bi001099p.
Binding pockets of the opioid receptors are presumably formed among the transmembrane domains (TMDs) and are accessible from the extracellular medium. In this study, we determined the sensitivity of binding of [(3)H]diprenorphine, an antagonist, to mu, delta, and kappa opioid receptors to charged methanethiosulfonate (MTS) derivatives and identified the cysteine residues within the TMDs that conferred the sensitivity. Incubation of the mu opioid receptor expressed in HEK293 cells with MTS ethylammonium (MTSEA), MTS ethyltrimethylammonium (MTSET), or MTS ethylsulfonate (MTSES) inhibited [(3)H]diprenorphine binding with the potency order of MTSEA > MTSET > MTSES. Pretreatment of mu, delta, and kappa opioid receptors with MTSEA dose-dependently inhibited [(3)H]diprenorphine binding with MTSEA sensitivity in the order of kappa > mu >> delta. The effects of MTSEA occurred rapidly, reaching the maximal inhibition in 10 min. (-)-Naloxone, but not (+)-naloxone, prevented the MTSEA effect, demonstrating that the reaction occurs within or in the vicinity of the binding pockets. Each cysteine residue in the TMDs of the three receptors was mutated singly, and the effects of MTSEA treatment were examined. The mutants had similar affinities for [(3)H]diprenorphine, and C7. 38(321)S, C7.38(303)S, and C7.38(315)S mutations rendered mu, delta, and kappa opioid receptors less sensitive to the effect of MTSEA, respectively. These results indicate that the conserved Cys7.38 is differentially accessible in the binding-site crevice of these receptors. The second extracellular loop of the kappa receptor, which contains several acidic residues, appears to play a role, albeit small, in its higher sensitivity to MTSEA, whereas the negative charge of Glu6.58(297) did not. To the best of our knowledge, this is the first report to show that a conserved residue among highly homologous G protein-coupled receptors is differentially accessible in the binding-site crevice. In addition, this represents the first successful generation of MTSEA-insensitive mutants of mu, delta, and kappa opioid receptors, which will allow determination of residues accessible in the binding-site crevices of these receptors by the substituted cysteine accessibility method.
阿片受体的结合口袋大概是在跨膜结构域（TMDs）之间形成的，并且可从细胞外介质进入。在本研究中，我们测定了拮抗剂[(3)H]二丙诺啡与μ、δ和κ阿片受体结合对带电荷的甲硫基磺酸盐（MTS）衍生物的敏感性，并确定了TMDs中赋予该敏感性的半胱氨酸残基。用MTS乙铵（MTSEA）、MTS乙基三甲基铵（MTSET）或MTS乙磺酸盐（MTSES）孵育HEK293细胞中表达的μ阿片受体，抑制[(3)H]二丙诺啡结合的效力顺序为MTSEA > MTSET > MTSES。用MTSEA对μ、δ和κ阿片受体进行预处理，剂量依赖性地抑制[(3)H]二丙诺啡结合，对MTSEA的敏感性顺序为κ > μ >> δ。MTSEA的作用迅速，在10分钟内达到最大抑制。(-)-纳洛酮而非(+)-纳洛酮可阻止MTSEA的作用，表明该反应发生在结合口袋内或其附近。对三种受体TMDs中的每个半胱氨酸残基进行单突变，并检测MTSEA处理的效果。这些突变体对[(3)H]二丙诺啡具有相似的亲和力，并且C7.38(321)S、C7.38(303)S和C7.38(315)S突变分别使μ、δ和κ阿片受体对MTSEA的作用敏感性降低。这些结果表明，保守的Cys7.38在这些受体的结合位点裂隙中的可及性不同。κ受体的第二个细胞外环含有几个酸性残基，尽管作用较小，但似乎在其对MTSEA的较高敏感性中起作用，而Glu6.58(297)的负电荷则不起作用。据我们所知，这是首次报道高度同源的G蛋白偶联受体中的一个保守残基在结合位点裂隙中的可及性不同。此外，这代表了首次成功构建对MTSEA不敏感的μ、δ和κ阿片受体突变体，这将允许通过取代半胱氨酸可及性方法确定这些受体结合位点裂隙中可及的残基。
