| During the past several years of my PhD studies, I strived to achieve a better understanding of the G-protein mediated phototransduction, particularly in a quantitative approach. Although the general picture of this signal transduction pathway has long been described, we lack accurate information about how individual step is precisely regulated. This work presents novel findings of phototransduction employing a combination of biochemical, electrophysiological, and genetic techniques.;As the major part of my research, we replaced cone transducin-alpha (cTalpha) for rod transducin-alpha (rTalpha) in transgenic mouse rods to evaluate how Galpha subtype influences signal amplification from different GPCRs, and how this might explain functional differences between rods and cones. We demonstrated biochemically that the exogenous cTalpha forms a functional heterotrimeric complex with rod Tbeta1gamma1. The electrophysiological recordings revealed that the light sensitivities were indeed affected by the expression level of G proteins and independent of cell-specific isoforms.;Taking advantages of variant G-protein expressing mice, we further investigated the relationship between transducin translocation pattern and its light activation. Because transducin translocation has been found to be triggered by saturation of the GTPase-activating complex that is proportional to light activation, we examined whether a higher light threshold to trigger translocation was required for a low expressor of transducin. In the cTalpha transgenic retina, we found out that light sensitivity appeared to be uncoupled from translocation threshold. Thus, additional factors other than transducin activation may contribute to its translocation.;The other part of my work aimed to study recoverin's influence on rhodopsin phosphorylation in the intact mouse rod photoreceptor cells. Consistent with many in vitro biochemical studies, our findings indicate Rv's inhibition on rhodopsin phosphorylation by quenching RK activity, which is likely to play an essential role during dark adaption. |
