Multiple Ligand Binding Sites of Rod and Cone Opsins

描述(申请人提供)：许多G蛋白偶联受体，包括视觉调节蛋白，在缺乏配体的情况下弱激活G蛋白。视蛋白与静息状态的反向激动剂11顺式视网膜的结合是独一无二的，11顺式视网膜在光的作用下会转化为激动剂。视杆蛋白与其11顺式视网膜生色团之间的相互作用在色素稳定性、活化和光谱调谐中的作用已被广泛研究。最近，有人提出视蛋白具有两个额外的视黄素结合位点，使视黄素能够接近和离开色素形成发色团结合口袋。在本研究中，我们将为OPTIN上的多个配体结合位点提供进一步的证据。更重要的是，我们假设除了维甲酸外，Opsins还与其他配体结合，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和光谱分析对溶液中的视紫红质进行实验，以检验三种功能。光调节通道容易受到维甲酸的强烈抑制。交替结合位点的一个功能可能是允许视紫红质和视蛋白作为缓冲，保护cGMP门控通道在暴露于强光后免受视黄醇的抑制，此时大量的视黄醇与色素上皮交换。视黄醇结合到交替位置的第二个功能是调节视紫红质和视蛋白的催化活性。这一功能可能在赋予单光子反应的重复性、设定受体的绝对灵敏度和反应动力学方面发挥作用。第三种功能可能是使化合物能够吸收光并将能量转移到视紫红质，导致视紫红质发生光异构化，并产生好像通过直接吸收一样的电响应。因此，可以在不改变视蛋白表达的情况下改变光谱敏感性。这些功能中的每一个都可以取决于视神经类型。光转导级联活性与视网膜疾病之间已经建立了很强的联系，这增加了影响交替配体结合位点或影响配体加工的突变可能是某些视网膜疾病的原因或贡献的可能性。然后，针对替代部位的药物可能被用于治疗视网膜疾病或增强视力。因此，这一建议对我们理解视觉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.