Characterization of the light activated properties of the novel photopigment melanopsin.

摘要

Many living organisms have biochemical and behavioral processes that are synchronized to the dark/light cycles of their environment. These processes have a diurnal rhythm to their activity, which shows a cycle of high and low activity within a 24hr period. Organisms must be able to adapt to daily and seasonal changes in light and photoperiod for survival. Timing of these events in mammals is regulated by cells in the suprachiasmatic nucleus (SCN) of the hypothalamus which make up the mammalian circadian clock. In mammals the circadian clock gets light information from the retina. In the retina there is a small class of intrinsically photosensitive retinal ganglion cells (ipRGCs) which express melanopsin photopigment and have axons that project directly to the SCN of the hypothalamus. In in vitro and in vivo experimental models, melanopsin can be expressed and reconstituted with 11-cis-retinal to form a functional photopigment that is able to activate a heterotrimeric G-protein in a light-dependent manner. The protein structure and the location of melanopsin expression in the retina suggest that it functions as the primary photopigment mediating light responses in ipRGCs. My research addresses the question of melanopsin's function by defining its characteristics as a light-activated photopigment in vitro. I hypothesized that melanopsin is a retinal photopigment receiving light input and transducing a biochemical signal that is important for photoentrainment in mammals. Using an in vitro biochemical approach to study the photochemical properties of heterologously expressed and endogenous melanopsin, I have demonstrated that melanopsin solubilized in detergent is sensitive to the ionic strength of the solution. Solubilized in a detergent solution with high or low NaCl concentration, heterologously expressed melanopsin forms two spectrally distinct photopigments with absorbance maxima of 420 nm or 480 nm, respectively. Surprisingly, when native melanopsin is purified from dark adapted mouse retinas the photopigment has a maximum absorbance of 500 nm. Finally, I present the first demonstration that native mouse melanopsin binds 11-cis retinal chromophore in the dark and light irradiation causes it to isomerize to all-trans retinal.