Modulation of Visual Transduction and Retinal Disease by Bicarbonate

描述(申请人提供)：视网膜杆和视锥通过水解cGMP发出光存在的信号，从而关闭质膜中的环核苷酸门控阳离子通道。一个向内的Na+电流被中断，细胞超极化。结果表明，碳酸氢钠能增加闪光响应幅度，加快响应动力学。然而，这种调节的许多特征尚不清楚，包括：作用的全部大小，剂量-反应关系，视网膜内碳酸氢盐的光感受器来源，碳酸氢盐进入外节的途径(S)，视杆细胞和视锥细胞之间的差异，以及碳酸氢盐的作用机制。使用单细胞记录和生化分析，我们将逐一解决这些问题。在阐明碳酸氢盐的作用机制时，我们将考虑三个靶点：鸟苷环化酶、PDE、环核苷酸门控通道。前两个目标将在生化分析中进行评估，而第三个目标将在体外膜片记录中进行测试。一些证据已经表明，小苏打可以刺激ROS-Gc1活性。然后，重要的是要找出ROS-GC2是否受到类似的影响，确定每个ROS-GC的碳酸氢盐结合部位，以及当钙敏感亚基GCAPs和S100B与ROS-GC结合时，确定碳酸氢盐刺激的钙依赖性。小苏打的视网膜来源将通过原位杂交和免疫组织化学进行定位。将阐明碳酸氢盐进入棒材并随后移除的途径，然后对其进行操作以表征碳酸氢盐对闪光的相对敏感度、闪光响应动力学和循环电流的影响。锥体在更明亮的光线下工作，具有更快的闪光响应动力学，并保持更高的代谢率。因此，重要的是要测试碳酸氢盐是否对锥体产生更大的影响，以及不同类型的锥体之间是否存在差异。完整小鼠的视网膜电信号记录将与单个视杆的记录进行比较，以确定现场的生理性碳酸氢盐水平。碳酸氢盐可以提供一种在持续暴露在强光下时防止饱和的方法，因此将探索对光适应的贡献。通过使突变小鼠处于高碳酸血症或抑制内源性碳酸氢盐的产生，将在突变小鼠身上测试碳酸氢盐对鸟苷环化酶活性的调节在加剧视网膜疾病中的作用。一个长期的目标是剖析在光和暗适应条件下塑造光子反应的分子机制，并了解它们在视网膜疾病中的作用，无论是起因还是调节。

DESCRIPTION (provided by applicant): Retinal rods and cones signal the presence of light by hydrolyzing cGMP thereby closing cyclic nucleotide gated cation channels in the plasma membrane. An inward Na+ current is interrupted and the cell hyperpolarizes. It has been shown that bicarbonate can increase the flash response amplitude and quicken response kinetics. However, many features of this modulation are not understood including: the full magnitudes of the effects, the dose-response relations, the intra-retinal sources of bicarbonate for photoreceptors, the pathway(s) for access of bicarbonate to the outer segment, differences between rods and cones, and the mechanism of bicarbonate action. Using single cell recording and biochemical assays, we will tackle each of these issues. In elucidating the mechanism of bicarbonate, we will consider three targets: guanylate cyclase, PDE, cyclic nucleotide gated channel. The first two targets will be evaluated in biochemical assays, while the third will be tested in excised membrane patch recordings. Some evidence already suggests that bicarbonate stimulates ROS-GC1 activity. It then becomes important to find out whether ROS-GC2 is similarly affected, to define the bicarbonate binding site of each ROS-GC, and to determine the calcium dependence of bicarbonate stimulation when the calcium sensing subunits GCAPs and S100B are bound to ROS-GCs. The retinal sources of bicarbonate will be localized with in situ hybridization and immunohistochemistry. Pathways for access of bicarbonate into rods and subsequent removal will be elucidated and then manipulated to characterize the impact of bicarbonate on relative sensitivity to flashes, flash response kinetics and the circulating current. Cones operate in brighter light, have quicker flash response kinetics and maintain a higher metabolic rate. It will therefore be important to test whether bicarbonate exerts a greater effect in cones and whether there are differences between various types of cones. Electroretinogram recordings from intact mice will be compared to recordings of single rods to define the physiological bicarbonate levels in situ. Bicarbonate could provide a means of preventing saturation during continuous exposure to bright light, so a contribution to light adaptation will be explored. A role for bicarbonate modulation of guanylate cyclase activity in exacerbating retinal disease will be tested in mutant mice by subjecting them to hypercapnia or by inhibiting endogenous bicarbonate production. A long term goal is to dissect the molecular mechanisms that shape the photon response under light and dark adapted conditions and to understand their roles, be it causative or modulatory, in retinal disease.