Function of the visual pigment in situ

描述(申请人提供)：脊椎动物光感受器的灵敏度和响应动力学取决于视觉色素分子的固有属性，如其光谱敏感性，以及影响视觉色素功能的环境因素，如视紫红质在盘膜中的横向迁移率。这项应用的目的是探索两个环境因素对光响应的影响：视紫红质堆积密度和转导蛋白的类型。参与光转导激活的前三个组分：视紫红质、转导蛋白和磷酸二酯酶，在盘膜表面相互作用。视紫红质激酶和RGS9这两个关闭光信号转导的关键蛋白也附着在膜上。在正常的棒中，光响应的激活和恢复中的限速步骤受到盘膜表面分子碰撞速率的限制。高的视紫红质堆积密度对膜蛋白迁移率的抑制作用减缓了这些速率，因为视紫红质堆积密度减少50%加速了光响应的上升和下降阶段。视紫红质堆积密度的进一步降低对光响应的影响不能可靠地预测，因为限速步骤的一致性可能会改变。堆积密度增加的影响的大小也无法预测，因为蛋白质迁移率随堆积密度的变化在体内是未知的。这些条件将在转基因小鼠的视紫红质表达棒中进行测试，这些转基因小鼠的视紫红质含量低于或超过正常水平。将通过吸电极记录棒对闪光和光阶跃的电反应来评估光转导。 杆状和锥状在灵敏度和响应动力学上有所不同。它们似乎具有相似的色素堆积密度，但总的来说，它们的色素和转导蛋白的身份是不同的。色素和转导蛋白类型对光响应灵敏度和动力学的影响将在火蜥蜴的视杆和视锥中进行测试，其中绿色视杆和蓝色感光视锥将相同的视觉色素与不同的转导蛋白偶联，而紫外线敏感视锥包含多种色素耦合到相同的转导蛋白。

DESCRIPTION (provided by applicant): The sensitivity and response kinetics of a vertebrate photoreceptor depend on the intrinsic properties of the visual pigment molecule, such as its spectral sensitivity, as well as environmental factors that affect visual pigment function, such as the lateral mobility of rhodopsin in the disk membrane. The goal of this application is to explore the impact of two environmental factors on the photoresponse: rhodopsin packing density and type of transducin present. The first three components involved in the activation of phototransduction: rhodopsin, transducin and phosphodiesterase, interact on the disk membrane surface. Rhodopsin kinase and RGS9, two key proteins in the shutoff of phototransduction are also attached to the membrane. In normal rods, the rate-limiting steps in the activation and recovery of the photoresponse are limited by the rates of molecular collisions on the disk membrane surface. These rates are slowed by the prohibitive effect of the high rhodopsin packing density on membrane protein mobility, because a 50% reduction in the rhodopsin packing density accelerated the rising and falling phases of the photoresponse. The effect of a further decrease in rhodopsin packing density on the photoresponse cannot be predicted reliably, because the identity of the rate-limiting step could change. The magnitude of the effect of an increase in packing density cannot be predicted either, because the change in protein mobility with packing density is not known in vivo. These conditions will be tested in rods of transgenic mice that express sub- and supranormal amounts of rhodopsin in their disk membranes. Phototransduction will be evaluated from suction electrode recordings of the electrical responses of the rods to flashes and steps of light. Rods and cones differ in their sensitivity and response kinetics. They appear to have similar pigment packing densities but in general, the identities of their pigments and transducins are different. The effects of pigment and transducin type on photoresponse sensitivity and kinetics will be tested in rods and cones of salamander, where green rods and blue-sensitive cones couple the same visual pigment to different transducins and UV-sensitive cones contain multiple pigments coupled to the same transducin.