Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, F-31077, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Institut de Pharmacologie et de Biologie Structurale, F-31077, Toulouse, France.
Innovations Thérapeutique et Résistance (Intheres). INTHERES, Université de Toulouse, INRA, ENVT, BP87614, F31076, Toulouse, Cedex 3, France.
Neurochem Int. 2020 Jan;132:104588. doi: 10.1016/j.neuint.2019.104588. Epub 2019 Nov 6.
This study compared pharmacological profiles between human mu opioid receptors (hMOR) overexpressed in the SH-SY5Y neuroblastoma cell line (SH-hMOR) and the methylotrophic yeast Pichia pastoris (Pp-hMOR). Affinity determinations were performed by direct binding with the tritiated agonist DAMGO and antagonist diprenorphine (DIP). Additionally, displacement of these drugs with agonists (morphine and DAMGO) and antagonists (β-funaltrexamine, naloxone and diprenorphine) was examined. Tritiated DAMGO could bind to membranes prepared from Pp-hMOR, although the receptor was not coupled with G-proteins. The data obtained with this yeast strain suggested that only 7.5% of receptors were in a high-affinity-state conformation. This value was markedly less than that estimated in SH-hMOR membranes, which reached 50%. Finally, to understand the pharmacological discrepancies between Pp-hMOR and SH-hMOR, the role of sterols was evaluated. The major sterol in P. pastoris is ergosterol, while hMOR naturally functions in a cholesterol-containing membrane environment. Cell membranes were sterol-depleted or cholesterol-loaded with methyl-β-cyclodextrine. The results indicated that cholesterol must be present to ensure Pp-hMOR function. The proportion of high-affinity-state conformation was reversibly increased by cholesterol complementation.
本研究比较了在 SH-SY5Y 神经母细胞瘤细胞系（SH-hMOR）和甲醇营养型酵母毕赤酵母（Pp-hMOR）中过表达的人 μ 阿片受体（hMOR）的药理学特征。通过与氚标记激动剂 DAMGO 和拮抗剂二苯并吗啡（DIP）的直接结合进行亲和力测定。此外，还检查了这些药物与激动剂（吗啡和 DAMGO）和拮抗剂（β-氟纳曲胺、纳洛酮和二苯并吗啡）的置换情况。氚标记的 DAMGO 可以与来自 Pp-hMOR 的膜结合，尽管该受体未与 G 蛋白偶联。从该酵母株获得的数据表明，只有 7.5%的受体处于高亲和力构象。这一数值明显低于在 SH-hMOR 膜中估计的 50%。最后，为了了解 Pp-hMOR 和 SH-hMOR 之间的药理学差异，评估了固醇的作用。毕赤酵母中的主要固醇是麦角固醇，而 hMOR 天然存在于含有胆固醇的膜环境中。用甲基-β-环糊精耗尽细胞或加载胆固醇细胞膜。结果表明，胆固醇的存在对于确保 Pp-hMOR 功能是必需的。胆固醇的补充可使高亲和力构象的比例可逆性增加。
