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

描述(申请人提供)：本研究项目的目标是揭示球果对光适应的分子机制。在任何给定的物种中，杆状对光的反应在时间进程中都比圆锥体反应慢，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光照的能力。圆锥体可以适应超过9个数量级的背景光强度，而杆状物体的适应能力有限。我们的假设认为，视锥细胞中的光适应部分是由于cGMP门控离子通道的配体敏感性的钙依赖变化引起的。在我们之前的工作中，我们表征了锥体调制，并观察到一种明显大于视杆的调制，这是由一种未知的可溶性调制蛋白介导的。最近，我们发现了一种新的蛋白，我们命名为CNG-调制素，它在锥体中表达，但不在杆状细胞中表达，并与锥体cGMP门控通道结合，同时在酵母和培养的细胞系中共表达。在初步实验中，这种蛋白质以一种钙离子依赖的方式调节通道对cGMP的敏感性。这些累积的观察结果强烈地表明，CNG-调制素是真正的锥体通道调节剂，但要明确分配这一功能，还需要进一步的功能研究。我们将通过以下实验来验证这一假设：(1)比较重组CNG-调制素在视锥细胞膜斑块中的调制与内源性调制的数量特征，以及(2)通过对转基因视锥细胞光感受器的功能研究来抑制CNG-调制素的表达。该项目的成功完成将首次验证锥形通道调制器的识别，并将使我们了解这一新的分子机制在锥体功能中的作用。它还将在光感受器相关遗传病的分子分析中提供另一种靶蛋白。 公共卫生相关性：该研究项目旨在表征一种新发现的锥体特异性蛋白的功能，该蛋白是锥体环核苷酸门控通道的调节器。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.