Modulation of Visual Transduction and Retinal Disease by Bicarbonate

碳酸氢盐对视觉传导和视网膜疾病的调节

• 批准号: 8759813
• 负责人: CLINT L MAKINO
• 金额: $ 40.63万
• 依托单位: MASSACHUSETTS EYE AND EAR INFIRMARY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8759813
• 关键词: Address; Affect; Aging; Attenuated; Behavior; Bicarbonates; Binding; Binding Sites; Biochemical; Biological Assay; Calcium; Carbon Dioxide; Carbonic Anhydrase Inhibitors; Catalytic Domain; Cell membrane; Cells; Complex; Cone; Cyclic GMP; Darkness; Defect; Dependence; Disclosure; Dose; Electrodes; Electroretinography; Excision; Exposure to; Goals; Guanylate Cyclase; Human; Hydrolysis; Hypercapnia; Immunohistochemistry; In Situ; In Situ Hybridization; Individual; Ion Channel; Kinetics; Lead; Light; Light Adaptations; Membrane; Metabolic; Metabolism; Modeling; Molecular; Multienzyme Complexes; Mus; Mutagenesis; Mutant Strains Mice; Mutation; Pathway interactions; Photons; Photoreceptors; Phototransduction; Physiological; Production; Recombinants; Relative (related person); Research; Retina; Retinal; Retinal Cone; Retinal Diseases; Retinal Photoreceptors; Rod Outer Segments; Role; Second Messenger Systems; Severities; Shapes; Signal Transduction; Source; Suction; Testing; Therapeutic Intervention; Vertebrate Photoreceptors; Visual; absorption; behavior change; carbonate dehydratase; cyclic nucleotide-gated cation channel; cyclic-nucleotide gated ion channels; design; extracellular; falls; in vitro testing; mutant mouse model; novel; prevent; public health relevance; research study; response; retinal rods; second messenger; therapeutic target

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.

描述（由申请人提供）：视网膜杆和锥体通过水解CGMP来信号，从而关闭质膜中的环状核苷酸门控阳离子通道。内向Na+电流中断，细胞超极化。已经表明，碳酸氢盐可以增加闪光反应幅度并加快响应动力学。但是，该调节的许多特征尚不理解包括：效果的完整幅度，剂量反应关系，碳酸氢盐的视网膜内源用于光感受器，碳酸氢盐访问外部片段的途径（S），杆和锥之间的差异，杆和锥之间的差异以及双碳酸盐剂的机制。使用单细胞记录和生化测定，我们将解决这些问题的每一个。在阐明碳酸氢盐的机理时，我们将考虑三个靶标：鸟苷酸环化酶，PDE，环状核苷酸门控通道。前两个目标将在生化测定中进行评估，而第三个目标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。然后，确定ROS-GC2是否受到类似影响，定义每个ROS-GC的碳酸氢盐结合位点，并确定当钙传感亚基GCAP和S100B绑定到ROS-GC时，这变得很重要。碳酸氢盐的视网膜来源将与原位杂交和免疫组织化学定位。将阐明碳酸氢盐进入杆的途径并随后的去除途径，然后操纵以表征碳酸氢盐对闪光灯，闪光反应动力学和循环电流的相对敏感性的影响。锥体以明亮的光线操作，具有更快的闪光反应动力学，并保持更高的代谢率。因此，测试碳酸氢盐是否在锥体上产生更大的作用以及各种锥体之间是否存在差异将很重要。将完整小鼠的电子图记录与单杆的记录进行比较，以定义原位生理碳酸氢盐水平。碳酸氢盐可以提供一种在连续暴露于明亮光期间预防饱和的方法，因此将探索对光适应的贡献。碳酸氢根环化酶活性在加剧视网膜疾病中调节的作用将在突变小鼠中测试，通过使它们患有高碳酸盐或抑制内源性碳酸氢盐的产生。一个长期的目标是剖析在视网膜疾病中塑造光子反应的分子机制，这些机制在光和深色适应条件下塑造了光子反应，并了解其作用，无论是病因还是调节性。