Multiple Ligand Binding Sites of Rod and Cone Opsins

描述（由申请人提供）：许多G蛋白偶联受体，包括视蛋白，在没有配体的情况下弱激活G蛋白。视蛋白的独特之处在于它们与静止状态下的逆激动剂11-顺式视网膜的结合，该受体通过光转化为激动剂。视杆蛋白与其11-顺式视网膜发色团之间的相互作用在色素稳定性、激活和光谱调谐方面的作用已经得到了很好的研究。最近，有人提出视蛋白具有两个额外的类视黄酮结合位点，使类视黄酮能够接近和退出色素形成的发色团结合袋。在本研究中，我们将进一步证明视蛋白上存在多个配体结合位点。更重要的是，我们假设视蛋白结合除了类维甲酸以外的其他配体，并且结合位点的功能超出了视觉周期。利用单细胞生理学、单细胞显微分光光度法和光谱学对溶液中的视紫红质进行了实验研究。光调节通道易受类维生素a的有效抑制。交替结合位点的一个功能可能是，当大量的类维甲酸与色素上皮交换时，视紫红质和视蛋白作为缓冲液，保护cgmp门控通道免受明亮光线下的类维甲酸抑制。类视黄醇与替代位点结合的第二个功能是调节视紫红质和视蛋白的催化活性。该函数可用于确定单光子响应的再现性、确定受体的绝对灵敏度和响应动力学。第三种功能可能是使化合物吸收光并将能量传递给视紫红质，使后者进行光异构化并产生类似于直接吸收的电反应。因此，光谱灵敏度可能在不改变视蛋白表达的情况下发生改变。这些功能中的每一个都可能取决于视蛋白类型。光传导级联活性与视网膜疾病之间已经建立了紧密的联系，这提出了影响替代配体结合位点或影响配体加工的突变可能导致或促成某些视网膜病变的可能性。然后，针对替代部位的药物可用于治疗视网膜疾病或增强视力。因此，这一发现对我们了解视视蛋白的结构和功能具有重要意义，并为视觉研究开辟了新的方向。

DESCRIPTION (provided by applicant): Many G protein coupled receptors, visual opsins included, weakly activate G protein in the absence of ligand. Opsins are unique in their binding of an inverse agonist at rest, 11-cis retinal, which is transformed into an agonist by light. The interactions between rod opsin and its 11-cis retinal chromophore have been well studied for their roles in pigment stability, activation and spectral tuning. Recently, it has been proposed that opsin possesses two additional retinoid binding sites that enable retinoids to approach and exit the pigment-forming chromophore binding pocket. In this study, we will provide further evidence for multiple ligand binding sites on opsin. More importantly, we hypothesize that opsins bind other ligands besides retinoids and that the functions of the binding sites extend beyond visual cycle. Using single cell physiology, single cell microspectrophotometry and spectroscopy on rhodopsin in solution, we propose experiments to test three functions. The light-regulated channel is susceptible to potent inhibition by retinoids. One function of the alternate binding sites may be to allow rhodopsin and opsin to serve as buffers that protect the cGMP-gated channel from retinoid inhibition after exposure to bright light, when vast amounts of retinoids are exchanged with the pigment epithelium. A second function of retinoid binding to alternate sites is to modulate the catalytic activity of rhodopsin and opsin. This function may play roles in conferring reproducibility to the single photon response, in setting the absolute sensitivity and response kinetics of the receptor. A third function may be to enable compounds to absorb light and transfer the energy to rhodopsin, causing the latter to photoisomerize and give rise to an electrical response as if by direct absorption. Thus spectral sensitivity may be altered without a change in opsin protein expression. Each of these functions may depend upon opsin type. A strong link has been established between phototransduction cascade activity and retinal disease raising the possibility that mutations affecting the alternate ligand binding sites or that affect ligand processing may be responsible for or contribute to certain retinal pathologies. Then pharmaceuticals that target the alternate site may be used for the treatment of a retinal disease or for the enhancement of vision. Therefore, this proposal will be highly significant to our understanding of the structure and function of visual opsins and opens new directions for vision research.