Tian He, Sakmar Thomas P, Huber Thomas
Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, NY.
Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, NY; Department of Neurobiology, Care Sciences and Society, Division of Neurogeriatrics, Center for Alzheimer Research, Karolinska Institutet, Huddinge, Sweden.
Biophys J. 2017 Jan 10;112(1):153-161. doi: 10.1016/j.bpj.2016.12.005.
The vertebrate visual photoreceptor rhodopsin (Rho) is a unique G protein-coupled receptor as it utilizes a covalently tethered inverse agonist (11-cis-retinal) as the native ligand. Previously, electrophysiological studies showed that ligand binding of 11-cis-retinal in dark-adapted Rho was essentially irreversible with a half-life estimated to be 420 years, until after thermal isomerization to all-trans-retinal, which then slowly dissociates. This long lifetime of 11-cis-retinal binding was considered to be physiologically important for minimizing background signal (dark noise) of the visual system. However, in vitro biochemical studies on the thermal stability of Rho showed that Rho decays with a half-life on the order of days. In this study, we resolve the discrepancy by measuring the chromophore exchange rate of the bound 11-cis-retinal chromophore with free 9-cis-retinal from Rho in an in vitro phospholipid/detergent bicelle system. We conclude that the thermal decay of Rho primarily proceeds through spontaneous breaking of the covalent linkage between opsin and 11-cis-retinal, which was overlooked in the electrophysiological recording. We estimate that this slow spontaneous release of 11-cis-retinal from Rho should result in 10 to 10 free opsin molecules in a dark-adapted rod cell-a number that is three orders of magnitude higher than previously expected. We also discuss the physiological implications of these findings on the basal activity of opsins and the associated dark noise in the visual system.
脊椎动物视觉光感受器视紫红质（Rho）是一种独特的G蛋白偶联受体，因为它利用共价连接的反向激动剂（11-顺式视黄醛）作为天然配体。此前，电生理研究表明，在暗适应的Rho中，11-顺式视黄醛的配体结合基本上是不可逆的，半衰期估计为420年，直到热异构化为全反式视黄醛后，全反式视黄醛才会缓慢解离。11-顺式视黄醛结合的这种长寿命被认为对最小化视觉系统的背景信号（暗噪声）具有重要生理意义。然而，关于Rho热稳定性的体外生化研究表明，Rho以数天的半衰期衰减。在本研究中，我们通过在体外磷脂/去污剂双分子层系统中测量结合的11-顺式视黄醛发色团与游离9-顺式视黄醛在Rho中的交换率，解决了这一差异。我们得出结论，Rho的热衰变主要通过视蛋白与11-顺式视黄醛之间共价键的自发断裂进行，而这在电生理记录中被忽略了。我们估计，11-顺式视黄醛从Rho的这种缓慢自发释放应该会在暗适应的视杆细胞中产生10到10个游离视蛋白分子，这个数字比之前预期的高三个数量级。我们还讨论了这些发现对视蛋白基础活性和视觉系统中相关暗噪声的生理影响。
