Signal Transduction in Retinal On-Bipolar Cells

描述（由申请人提供）：本提案中描述的实验旨在了解脊椎动物视网膜上双极细胞突触的光感受器。双极细胞使用一种g蛋白偶联的信号机制，将它从光感受器收集的信息传递到视网膜内部进行处理。虽然受体和g蛋白已被确定，但将它们与非特异性阳离子通道联系起来的机制尚不清楚。我将用以下策略来研究这个级联。在阻断g蛋白作用的同时，我将确定在双极细胞中需要哪些内部因子来打开转导通道。要做到这一点，我将使整个细胞膜片钳记录从蝾螈双极细胞。特别是，我将测试转导通道的天然状态是打开还是关闭，以及门控机制是否直接或间接涉及cGMP。如果不涉及cGMP，则将确定通道打开的代谢需求。在平行实验中，我将确定哪些内部因素是维持光响应所必需的，或者哪些因素是通道关闭所必需的。我还将测试利用非特异性阳离子通道的其他已知信号机制的抑制剂。这两种类型的实验将通过钙在双极枝上的双光子成像来证实，以比较“跑开”通道和那些由转导机制打开的通道的定位。最后，我将研究如何在小鼠视网膜杆状光感受器和杆状双极细胞之间传递低光水平的信息。该突触的信噪比通过阈值非线性最大化。我将研究产生这种非线性的机理。首先，我将确定该机制是突触前还是突触后，以及它是由受体饱和还是下游转导机制引起的。最后，我将确定非线性是否是动态的，或者它的位置是否会受到非常暗淡的背景光的影响。

DESCRIPTION (provided by applicant): The experiments described in this proposal are aimed understanding the photoreceptor to On-bipolar cell synapse in the vertebrate retina. The On-bipolar cell uses a G-protein-coupled signaling mechanism to relay information it collects from the photoreceptors to the inner retina for processing. While the receptor and G-protein have been identified, the mechanisms linking these to a non-specific cation channel are unknown. I will approach the study of this cascade with the following strategy. While blocking the G-protein's action, I will determine what internal factors are necessary in On-bipolar cells to open transduction channels. To accomplish this I will make whole-cell patch clamp recordings from salamander On-bipolar cells. In particular I will test whether the native state of the transduction channel is open or closed, and whether gating mechanisms directly or indirectly involve cGMP. If cGMP is not involved, the metabolic requirements for channel opening will be identified. In parallel experiments I will determine what internal factors are necessary for maintaining the light response, or what factors are necessary for channel closure. I will also test inhibitors of other known signaling mechanisms that utilize non-specific cation channels. These two types of experiments will be corroborated by two-photon imaging of calcium in the On-bipolar dendrites, to compare the localization of "run-open" channels with those opened by the transduction mechanism. Finally, I will study how low light level information is transferred between rod photoreceptors and rod bipolar cells in the mouse retina. The signal-to-noise ratio at this synapse is maximized by a thresholding non-linearity. I will study the mechanism producing this non-linearity. First I will determine whether the mechanism is pre- or postsynaptic, and if it arises from saturation at the receptors or downstream in the transduction mechanism. Lastly, I will determine whether the non-linearity is dynamic, or whether its position can be influenced by very dim background light.