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理解脊椎动物视网膜中的光感受器与双极细胞突触。双极细胞使用G蛋白偶联信号机制将其从光感受器收集的信息传递到内层视网膜进行处理。虽然受体和G蛋白已被鉴定，但将它们与非特异性阳离子通道连接的机制尚不清楚。我将采用以下策略来研究这一级联。在阻断G蛋白的作用时，我将确定在双极细胞中打开转导通道所需的内部因素。为了实现这一点，我将对蝾螈双极细胞进行全细胞膜片钳记录。特别是，我将测试转导通道的天然状态是开放还是关闭，以及门控机制是否直接或间接涉及cGMP。如果不涉及cGMP，则将确定通道开放的代谢要求。在平行实验中，我将确定维持光响应所需的内部因素，或者通道关闭所需的因素。我还将测试其他已知的利用非特异性阳离子通道的信号传导机制的抑制剂。这两种类型的实验将通过双光子成像的钙在双极树突，比较本地化的“运行开放”通道与那些打开的转导机制。最后，我将研究如何低光水平的信息传递之间的杆光感受器和杆双极细胞在小鼠视网膜。通过阈值非线性使该突触处的信噪比最大化。我将研究产生这种非线性的机制。首先，我将确定这种机制是突触前还是突触后，以及它是由受体饱和还是转导机制下游引起的。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昏暗的背景光的影响。

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.