Function of the visual pigment in situ

描述（由申请人提供）：脊椎动物光感受器的灵敏度和响应动力学取决于视色素分子的内在特性，如其光谱灵敏度，以及影响视色素功能的环境因素，如视紫红质在盘膜中的横向迁移。本应用的目的是探讨两个环境因素对光响应的影响：视紫红质堆积密度和存在的转导素类型。参与光导激活的前三种成分：视紫红质、转导素和磷酸二酯酶，在盘膜表面相互作用。Rhodopsin kinase和RGS9这两个关闭光转导的关键蛋白也附着在膜上。在正常的棒中，激活和恢复光响应的速率限制步骤受到磁盘膜表面分子碰撞速率的限制。高视紫红质包装密度对膜蛋白迁移的抑制作用减慢了这些速率，因为视紫红质包装密度降低50%会加速光响应的上升和下降阶段。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，因为限速步骤的身份可能会改变。增加包装密度的影响程度也无法预测，因为蛋白质迁移率随包装密度的变化在体内是未知的。这些条件将在转基因小鼠棒中进行测试，这些小鼠在其盘膜中表达亚正常和超正常的视紫红质量。光导将通过吸电极记录杆状体对闪光和光线的电反应来评估。

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.