The elongated cilia of the outer segment of rod and cone photoreceptor cells can contain concentrations of visual pigments of up to 5 mM. The rod visual pigments, G protein-coupled receptors called rhodopsins, have a propensity to self-aggregate, a property conserved among many G protein-coupled receptors. However, the effect of rhodopsin oligomerization on G protein signaling in native cells is less clear. Here, we address this gap in knowledge by studying rod phototransduction. As the rod outer segment is known to adjust its size proportionally to overexpression or reduction of rhodopsin expression, genetic perturbation of rhodopsin cannot be used to resolve this question. Therefore, we turned to high-throughput screening of a diverse library of 50,000 small molecules and used a novel assay for the detection of rhodopsin dimerization. This screen identified nine small molecules that either disrupted or enhanced rhodopsin dimer contacts in vitro. In a subsequent cell-free binding study, we found that all nine compounds decreased intrinsic fluorescence without affecting the overall UV-visible spectrum of rhodopsin, supporting their actions as allosteric modulators. Furthermore, ex vivo electrophysiological recordings revealed that a disruptive, hit compound #7 significantly slowed down the light response kinetics of intact rods, whereas compound #1, an enhancing hit candidate, did not substantially affect the photoresponse kinetics but did cause a significant reduction in light sensitivity. This study provides a monitoring tool for future investigation of the rhodopsin signaling cascade and reports the discovery of new allosteric modulators of rhodopsin dimerization that can also alter rod photoreceptor physiology.

中文翻译：

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鉴定作为视紫红质寡聚化增强剂/破坏剂的小分子变构调节剂。

视杆细胞和视锥细胞外段的细长纤毛可含有浓度高达 5 mM 的视觉色素。视杆视觉色素，称为视紫红质的 G 蛋白偶联受体，具有自我聚集的倾向，这是许多 G 蛋白偶联受体中保守的特性。然而，视紫红质寡聚化对天然细胞中 G 蛋白信号传导的影响尚不清楚。在这里，我们通过研究杆状光转导来解决这一知识差距。由于已知杆外段会根据视紫红质表达的过度表达或减少成比例地调整其大小，因此不能使用视紫红质的遗传扰动来解决这个问题。因此，我们转向对包含 50,000 个小分子的多样化文库进行高通量筛选，并使用一种新的检测方法来检测视紫红质二聚化。该筛选确定了九个在体外破坏或增强视紫红质二聚体接触的小分子。在随后的无细胞结合研究中，我们发现所有九种化合物都降低了内在荧光而不影响视紫红质的整体紫外可见光谱，支持它们作为变构调节剂的作用。此外，离体电生理记录显示，破坏性的命中化合物#7 显着减慢了完整视杆的光响应动力学，而增强的命中候选化合物#1 并没有显着影响光响应动力学，但确实导致光响应动力学显着降低。光敏感度。