Modulation of Visual Transduction and Retinal Disease by Bicarbonate

摘要 视网膜视杆和视锥通过降解cGMP发出光存在的信号，cGMP随后关闭循环 质膜上的核苷酸门控阳离子通道。内向的Na+电流被中断，细胞 超极化。结果表明，碳酸氢钠能增加闪光反应幅度，加快闪光反应速度。 响应动力学。然而，这种调制的许多特征没有被理解，包括：完整的 影响的大小，剂量-反应关系，视网膜内碳酸氢盐的来源 光感受器，小苏打进入外节的途径(S)，杆之间的差异 和圆锥体，以及重碳酸盐的作用机制。使用单细胞记录和生化分析， 我们会逐一处理这些问题。棒的外段有一个阴离子交换器，通常 会挤出小苏打。但通过去除细胞外的氯离子，我们可以迫使阴离子交换器在杆的外部 以吸收重碳酸盐的片段。然后我们将描述碳酸氢盐是如何影响幅度和动力学的 在膜电流的单细胞记录中，明亮的闪光和暗淡的闪光响应。明亮的， 饱和闪光响应提供了循环电流的测量。响应动力学因闪光而异 闪光强度不变，但闪光强度非常低，因此暗淡的闪光响应对分析很有用 反应动力学。为了找出碳酸氢盐在以下情况下通常如何接触棒材外段 细胞外氯离子的存在，我们将改变记录配置：吸管内的外段VS 移液管内的内段，孤立的视杆与视杆连接在一块视网膜上。我们将探索 应用碳酸氢酶调节内源碳酸氢盐对球果光信号转导的调节 当记录时从分离的视锥细胞和附着在视网膜碎片上的视锥细胞开始阐明 动态调制发挥作用的条件。为了充分描述碳酸氢盐对 为了与棒的结果进行比较，我们将使用外源碳酸氢盐进行实验， 与提议的棒材类似，只是将存在碳酸酐酶。在阐明 碳酸氢盐的作用机制，我们将考虑三个靶点：鸟苷环化酶、PDE、环核苷酸门控 频道。前两个目标将在生化分析中进行评估，而第三个目标将在 切除的膜贴片记录。一些证据已经表明，重碳酸盐刺激ROS- Gc1活动。然后，重要的是找出ROS-GC2是否受到类似的影响，以定义 每种ROS-GC的小苏打结合部位，并测定小苏打对钙的依赖性 当钙敏感亚基GCAPs和S100B与ROS-GCs结合时的刺激。长期的 目的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。

SUMMARY Retinal rods and cones signal the presence of light by hydrolyzing cGMP which then closes 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. Rods have an anion exchanger in their outer segments that normally extrudes bicarbonate. But by removing extracellular Cl-, we can force the anion exchanger in rod outer segments to take up bicarbonate. We will then describe how bicarbonate affects the amplitude and kinetics of both bright flash and dim flash responses in single cell recordings of membrane current. The bright, saturating flash response provides a measure of the circulating current. Responses kinetics vary with flash strength but become invariant with very low flash strengths, so the dim flash response is useful for analysis of response kinetics. To find out how bicarbonate normally accesses the rod outer segment when extracellular Cl- is present, we will change the recording configuration: outer segment inside the pipette vs inner segment inside the pipette, isolated rod vs rods attached to a piece of retina. We will explore endogenous bicarbonate modulation of phototransduction in cones by applying a carbonic anhydrase inhibitor while recording from isolated cones and cones attached to pieces of retina and begin to elucidate conditions in which dynamic modulation comes into play. To fully characterize the effects of bicarbonate on cones for comparison to results from rods, we will carry out experiments with exogenous bicarbonate, similar to those proposed for rods except that a carbonic anhydrase will be present. 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. 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.