Investigation of Inactive-State κ Opioid Receptor Homodimerization via Single-Molecule Microscopy Using New Antagonistic Fluorescent Probes

Opioid receptors (ORs) are among the best-studied G protein-coupled receptors due to their involvement in neurological disorders and important role in pain treatment. Contrary to the classical monomeric model, indirect evidence suggests that ORs might form dimers, which could be endowed with a distinct pharmacological profile, and, thus, be targeted to develop innovative pharmacological therapies. However, direct evidence for the spontaneous formation of OR dimers in living cells under physiological conditions is missing. Despite a growing interest in the κ opioid receptor (KOR), KOR-selective fluorescent probes are particularly scarce in the literature. Herein, we present the first set of fluorescent KOR-selective probes with antagonistic properties. Two of these were employed in single-molecule microscopy (SMM) experiments to investigate KOR homodimerization, localization, and trafficking. Our findings indicate that most KORs labeled with the new fluorescent probes are present as apparently freely diffusing monomers on the surface of a simple cell model.阿片受体（Opioid receptors, ORs）是研究最为深入的G蛋白偶联受体（G protein-coupled receptors）之一，因其参与神经系统疾病的发生发展并在疼痛治疗中发挥关键作用而广受关注。与经典的单体模型相悖，间接证据表明阿片受体可能形成二聚体，这类二聚体具备独特的药理学特征，因此可作为开发创新性药理学疗法的靶点。然而，目前仍缺乏生理条件下活细胞中阿片受体二聚体自发形成的直接实验证据。尽管学界对κ阿片受体（κ opioid receptor, KOR）的研究兴趣日益高涨，但文献中选择性靶向KOR的荧光探针仍极为稀缺。本研究首次报道了一系列具备拮抗活性的选择性KOR荧光探针。我们选取其中两款探针开展单分子显微镜（single-molecule microscopy, SMM）实验，以探究KOR的同源二聚化、定位及膜转运过程。研究结果显示，经新型荧光探针标记的KOR大多以可自由扩散的单体形式存在于简化细胞模型的细胞膜表面。