Modulation of Visual Transduction and Retinal Disease by Bicarbonate

描述（由申请人提供）：视网膜杆状细胞和视锥细胞通过水解cGMP来发出光的信号，从而关闭质膜中的环核苷酸门控阳离子通道。向内的Na+电流中断，细胞发生超极化。研究表明，碳酸氢盐能提高闪变响应幅度，加快闪变响应动力学。然而，这种调节的许多特征尚不清楚，包括：影响的全部幅度、剂量-反应关系、光感受器中碳酸氢盐的视网膜内来源、碳酸氢盐进入外段的途径、视杆细胞和视锥细胞之间的差异以及碳酸氢盐作用的机制。使用单细胞记录和生化分析，我们将解决这些问题。在阐明碳酸氢盐的作用机制时，我们将考虑三个靶点：鸟苷酸环化酶、PDE、环核苷酸门控通道。前两个靶点将在生化分析中进行评估，而第三个靶点将在切除的膜贴片记录中进行测试。一些证据已经表明，碳酸氢盐刺激ROS-GC1活性。因此，确定ROS-GC2是否受到类似的影响，确定每种ROS-GC的碳酸氢盐结合位点，以及确定钙敏感亚基GCAPs和S100B与ROS-GC结合时碳酸氢盐刺激对钙的依赖性变得非常重要。用原位杂交和免疫组织化学方法定位视网膜中碳酸氢盐的来源。将阐明碳酸氢盐进入棒和随后去除的途径，然后进行操作，以表征碳酸氢盐对闪光的相对灵敏度、闪光响应动力学和循环电流的影响。视锥细胞在更明亮的光线下工作，具有更快的闪光反应动力学，并保持更高的代谢率。因此，测试碳酸氢盐是否对视锥细胞产生更大的影响以及不同类型的视锥细胞之间是否存在差异将是很重要的。完整小鼠的视网膜电图记录将与单杆的记录进行比较，以确定原位生理碳酸氢盐水平。碳酸氢盐可以在连续暴露在强光下时提供防止饱和的手段，因此将探讨对光适应的贡献。将在突变小鼠中通过高碳酸血症或抑制内源性碳酸氢盐的产生来测试碳酸氢盐对鸟苷酸环化酶活性的调节在加剧视网膜疾病中的作用。长期目标是剖析在光和暗适应条件下形成光子响应的分子机制，并了解它们在视网膜疾病中的作用，无论是致病的还是调节的。

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.