Identity of a cone-specific cGMP-gated Channel Modulator

描述（由申请人提供）：杆状光感受器和锥状光感受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，杆状体几乎不适应，在非哺乳动物物种中，它们在一定程度上是适应的。相比之下，所有脊椎动物的视锥细胞都能适应绝对强度在10个对数单位范围内变化的背景光。本研究项目的长期目标是揭示锥体光感受器光适应的分子机制。最近的研究表明，视杆细胞和视锥细胞之间最显著的差异反映了一种尚未确定的蛋白质的功能，该蛋白质以Ca2+依赖的方式调节视锥细胞cGMP门控通道对cGMP的敏感性。这个功能在杆中根本不存在。为了继续这项研究，必须对锥形信道调制器进行识别和表征。这一目标将通过分子遗传学方法实现，具体目标如下：鉴定锥体光感受器环gmp门控离子通道的Ca2+依赖性调节剂。酵母双杂交筛选将通过其与cgmp门控通道细胞质域的相互作用来鉴定调节剂。具体目标2。验证特异性Aim I蛋白在体外与通道的物理相互作用以及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。具体目标3。测试克隆调制器的功能有效性。克隆调制器的功能特性将在完整的视锥细胞中进行电生理实验。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Ca2+依赖性调节cGMP门控通道配体敏感性的新分子机制，这是锥体光感受器的特征，而不存在于杆状体中。成功将为光感受器相关遗传疾病（例如视网膜色素变性）的分子分析提供另一种靶蛋白。

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).