My long-term goal is to understand intracellular signaling cascades and their contribution to image processing in retina. As the eye scans a scene, each photoreceptor sees alternating pulses of light and dark that decrease or increase its glutamate release. Each burst of glutamate has two effects: to directly open an AMPA/kainate cation channel in OFF bipolar cells, and to indirectly close an unidentified cation channel in ON bipolar cells. The first step toward channel closure is activation of the G-protein Go1 by the metabotropic receptor mGluR6, but the rest of the cascade is largely unknown. Our goal is to elucidate the full cascade, its regulators, and the channel. We have now identified two strong interactors of Galphao1, Ret-RGS1 and Pcp2. Both colocalize with mGluR6 and Galphao1, coimmunoprecipitate with Galphao1, and modulate G(o1's activity in vitro. Ret-RGS1 catalyzes Galphao's GTPase activity (to deactivate Go), and Pcp2 is thought to inhibit GDP dissociation from Galphao (to slow Go's activation). Because the light response arises when Go is deactivated, and terminates when Go is activated, Ret-RGS1 is hypothesized to accelerate the rising phase of the light response and Pcp2 to prolong its falling phase. To test these hypotheses AIMS 1 and 2 will: (1) test effect of Pcp2 in oocytes expressing the mGluR6 transduction elements; (2) record the dynamics of the electroretinogram b-wave from Pcp2- and ret-RGS1-null mice; and (3) record direct responses from ON bipolar cells of these null mice to puffs of mGluR6 antagonist (whole cell configuration).
AIM 3 will identify the ON bipolar transduction channel. This will be accomplished by expression cloning and hybridization using an ON bipolar cDNA library. We have already produced a transgenic mouse with EGFP-expressing ON bipolar cells and have isolated these cells. The isolated cells have been used to construct an ON bipolar cDNA library. The library will be expressed with mGluR6 and G(o1 in Xenopus oocytes, and responses to mGluR6 agonist will be tested.
AIM 4 will characterize the channel's fundamental biophysical properties, such as its gating molecules, voltage dependence, sensitivity to Ca2+, and effect of phosphorylation. This effort will contribute fundamental understanding of the first synapse on the visual pathway and of night blindness; it should thus extend the basic foundation for future clinical studies.
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