Function of CNG-modulin, a novel regulator of the cone cGMP-gated channel

描述（由申请人提供）：本研究项目的目的是揭示负责锥细胞光适应的分子机制。在任何给定的物种中，视杆细胞对光的反应在时间过程上都比视锥细胞反应慢，并且对光更敏感。但视杆细胞和视锥细胞之间最显著的区别在于它们适应环境照明的能力。视锥细胞可以适应超过9个数量级的背景光强度，而视杆细胞的适应能力有限。我们的假设指出，光适应锥产生，部分，从Ca 2+依赖性变化的cGMP门控离子通道的配体敏感性。在我们以前的工作中，我们的特点锥调制，并观察到一个显着更大的调制比杆，这是由一个未知的可溶性调节蛋白介导的。最近，我们已经确定了一种新的蛋白质，我们命名为CNG-modulin，这是在锥中表达，但不是在杆，并结合到锥cGMP门控通道，同时在酵母和培养的细胞系中共表达。 在试点 在实验中，这种蛋白质以Ca 2+依赖性方式调节来自条纹低音锥的切除膜斑中通道对cGMP的敏感性。这些累积的观察结果强烈表明，CNG-modulin是真实的锥通道调制器，但需要进一步的功能研究，明确分配此功能。我们将测试这一假设与实验设计（1）比较的调制的定量特征，使用重组CNG-调制蛋白的锥膜补丁与内源性调制，（2）通过功能研究的基因修饰的锥光感受器改变，以抑制CNG-调制蛋白的表达。该项目的成功完成将首次验证锥通道调制器的鉴定，并将为我们提供对这种新型分子机制在锥功能中的作用的理解。它也将为光感受器相关遗传疾病的分子分析提供另一个靶蛋白。 公共卫生相关性： 本研究旨在探讨一种新发现的视锥细胞特异性蛋白的功能，它是视锥细胞环核苷酸门控通道的调节剂。这将进一步加深我们对光适应的分子机制的理解，并将为探索其在光感受器相关遗传疾病中的可能作用提供基础。

DESCRIPTION (provided by applicant): The objective of this research project is to uncover molecular mechanisms responsible for light adaptation in cones. In any given species, rod responses to light are slower in time course and more sensitive to light than cone responses. But the most dramatic difference between rods and cones is in their ability to adapt to ambient illumination. Cones can adapt to over 9 orders of magnitude of the background light intensity, while rods have a limited ability to adapt. Our hypothesis states that light adaptation in cones arises, in part, from Ca2+dependent changes in the ligand sensitivity of the cGMP-gated ion channels. In our previous work, we characterized cone modulation and observed a significantly larger modulation than rods, which is mediated by an unknown soluble modulator protein. Recently, we have identified a novel protein that we named CNG-modulin, which is expressed in cones, but not in rods, and binds to the cone cGMP-gated channel while co-expressed in yeast and in cultured cell lines. In pilot experiments, this protein modulates the sensitivity of the channel to cGMP in a Ca2+dependent manner in excised membrane patches from striped bass cones. These cumulative observations strongly suggest that CNG-modulin is the authentic cone channel modulator, but further functional studies are required for an unambiguous assignment of this function. We will test this hypothesis with experiments designed (1) to compare the quantitative features of the modulation using recombinant CNG-modulin in cone membrane patches with those of the endogenous modulation, and (2) through functional studies of genetically modified cone photoreceptors altered to suppress the expression of CNG-modulin. Successful completion of this project will verify, for the first time, the identification of the cone channel modulator and will provide us with the understanding of the role of this novel molecular mechanism in the cone function. It will also provide another target protein in the molecular analysis of photoreceptor-related genetic diseases. PUBLIC HEALTH RELEVANCE: This research project aims to characterize the function of a newly discovered cone- specific protein, a modulator of the cone cyclic nucleotide gated channel. It will further our understanding of the molecular mechanism of light adaptation and will provide a basis for exploring its possible role in photoreceptor-related genetic diseases.