Identity of a cone-specific CGMP-gated Channel Modulator

描述(申请人提供)：视杆感光器和视锥感光器之间最显著的区别是它们适应背景照明的能力。在哺乳动物中，杆状物几乎不适应，在非哺乳动物物种中，它们在一定程度上适应。相比之下，所有脊椎动物的锥体都适应在大约10个对数单位的绝对强度范围内变化的背景光。该研究项目的长期目标是揭示锥体光感受器中光适应的分子机制。最近的研究表明，视杆细胞和视锥细胞之间的这种最显著的差异反映了一种尚未确定的蛋白的功能，该蛋白以钙离子依赖的方式调节视锥细胞cGMP门控通道对cGMP的敏感性。这种功能根本不存在于棒中。为了继续这项研究，必须识别和表征锥形通道调制器。这一目标将通过分子遗传学的方法来实现，具体目的如下：1.鉴定视锥细胞光感受器环状GMP门控离子通道的钙依赖调节剂。酵母双杂交筛选将通过其与cGMP门控通道胞质结构域的相互作用来鉴定该调节子。特定目的2.验证特定目的I蛋白在体外与通道的物理相互作用，以及在原位相互作用的生理相关性。体外结合试验、原位杂交和免疫组织化学将用于验证相互作用的生理相关性。具体目的3.检测克隆调控子的功能有效性。克隆的调节器的功能特性将在电生理实验中在完整的视锥细胞中进行测试。如果成功，这种以前未被识别的蛋白质的发现和分析将对阐明一个长期存在的生理谜团产生影响。这个项目的成功完成将使我们第一次详细了解cGMP门控通道配体敏感性的钙依赖调节的新的分子机制，这是视锥体光感受器所特有的，在视杆细胞中不存在。这项研究的成功将为与光感受器相关的遗传性疾病(例如视网膜色素变性)的分子分析提供另一种目标蛋白。

DESCRIPTION (provided by applicant): The most dramatic difference between rod and cone photoreceptors is their ability to adapt to background illumination. In mammals, rods hardly adapt at all and they do to some limited extent in non-mammalian species. In contrast, cones in all vertebrate species adapt to background lights that change over a range of about 10 log units of absolute intensity. The long-term objective of this research project is to uncover the molecular mechanisms responsible for light-adaptation in cone photoreceptors. It was recently demonstrated that this most significant difference between rods and cones reflects the function of a yet to be identified protein that modulates the sensitivity to cGMP of the cGMP-gated channels of cones in a Ca2+-dependent manner. This function simply does not exist in rods. To continue this investigation, the cone channel modulator must be identified and characterized. This objective will be followed through molecular genetics approach with the following specific aims: Specific Aim 1. To identify the Ca2+ dependent modulator of the cyclic GMP-gated ion channels of cone photoreceptors. Yeast two-hybrid screening will be used to identify the modulator by its interaction with the cGMP-gated channel cytoplasmic domain. Specific Aim 2. To verify the physical interactions of the identified under Specific Aim I protein with the channel in vitro and the physiological relevance of the interaction in situ. In vitro binding assays, in situ hybridization and immunohistochemistry will be used to verify the physiological relevance of the interactions. Specific Aim 3. Test the functional effectiveness of cloned modulator. The functional characteristics of the cloned modulator will be tested in intact cones in electrophysiological experiments. If successful, discovery and analysis of this previously unrecognized protein would be of impact in the elucidation of a long-standing physiological enigma. The successful completion of this project will provide us for the first time with detailed understanding of a novel molecular mechanism of the Ca2+-dependent modulation of cGMP gated channel ligand sensitivity that is characteristic for cone photoreceptors and is not present in rods. Success will provide yet another target protein in the molecular analysis of photoreceptorrelated genetic diseases (retinitis pigmentosa, for example).