Boyer Nicholas P, Chen Chunhe, Koutalos Yiannis
Storm Eye Institute, Medical University of South Carolina.
J Vis Exp. 2011 Jun 22(52):2789. doi: 10.3791/2789.
In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells¹⁻⁴. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca²+ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca²+ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light. Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the photoisomerization of its 11-cis chromophore to all-trans⁵⁻⁷. This regeneration begins with the release of all-trans retinal by the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all- trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP). Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca²+-sensitive fluorescent dyes can be used to examine in detail the interplay between outer segment Ca²+ changes and response to light⁸⁻¹² as well as the role of inner segment Ca²+ stores in Ca²+ homeostasis¹³⁻¹⁴. Fluorescent dyes can also be used for measuring Mg² concentration¹⁵, pH, and as tracers of aqueous and membrane compartments¹⁶. Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single photoreceptor cells¹⁷⁻¹⁹.
在脊椎动物视网膜中，光转导（即光转化为电信号的过程）由视杆和视锥光感受器细胞完成¹⁻⁴。视杆光感受器负责暗光下的视觉，视锥光感受器负责亮光下的视觉。光转导发生在光感受器细胞的外段，这是一个特殊的区域，含有高浓度的视觉色素，即主要的光探测器。视觉色素由一个发色团（11-顺式视黄醛）与一种蛋白质（视蛋白）结合而成。视觉色素吸收的一个光子会使发色团从11-顺式异构化为全反式。这种光异构化会导致视觉色素发生构象变化，引发一系列反应，最终导致膜电位改变，从而将光刺激转化为电信号。细胞从光刺激中恢复涉及光激活的中间体失活以及膜电位的重新建立。Ca²⁺调节参与光转导的几种酶的活性，并且在光刺激时其浓度会降低。通过这种方式，Ca²⁺在细胞从光刺激中恢复及其对背景光的适应过程中发挥重要作用。恢复过程的另一个重要部分是视觉色素的再生，其11-顺式发色团在光检测过程中通过光异构化转化为全反式而被破坏⁵⁻⁷。这种再生始于光激活的色素释放全反式视黄醛，留下脱辅基蛋白视蛋白。释放的全反式视黄醛在利用NADPH的反应中迅速还原为全反式视黄醇，视蛋白与进入外段的新鲜11-顺式视黄醛结合，重新形成视觉色素。然后，全反式视黄醇通过特殊载体细胞间视黄醛结合蛋白（IRBP）从外段转运到相邻细胞中。单个光感受器细胞的荧光成像可用于研究其生理学和细胞生物学。Ca²⁺敏感的荧光染料可用于详细研究外段Ca²⁺变化与光反应之间的相互作用⁸⁻¹²，以及内段Ca²⁺储存库在Ca²⁺稳态中的作用¹³⁻¹⁴。荧光染料还可用于测量Mg²⁺浓度¹⁵、pH值，以及作为水相和膜相区室的示踪剂¹⁶。最后，全反式视黄醇（维生素A）的固有荧光可用于监测其在单个光感受器细胞中的形成和去除动力学¹⁷⁻¹⁹。
