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M.T. Walker, P.R. Robinson; Characterization of the Light Activated Properties of the Novel Photopigment Melanopsin . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1724.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: It has recently been shown that rod and cone photoreceptors are not necessary for circadian entrainment in mammals. There is a subset of retinal ganglion cells (RGCs) in the inner retina of mammals that are intrinsically photosensitive. This subset of RGCs have axons that project directly to the suprachiasmatic nucleus, and they express the opsin–like protein melanopsin. Melanopsin has been suggested to function as a photopigment in these RGCs. Electrophysiological recordings from melanopsin expressing RGCs demonstrate a maximum sensitivity to 484nm light. In previous work from our laboratory, we have been able to express melanopsin in vitro and demonstrate that the expressed protein can be reconstituted with 11–cis retinal and form a functional photopigment with a maximum wavelength sensitivity of 424nm. There is a ∼60nm difference between the wavelength sensitivity of the intrinsic light response of RGCs and the expressed melanopsin photopigment. We are interested in whether conditions in the in vitro assay could contribute to this large spectral difference. In addition, we are interested in the conditions of the chromophore binding pocket that might contribute to these spectral differences. To study these questions, we have examined the affect of pH and salinity on the spectral sensitivity of expressed melanopsin. We have also used site directed mutagenesis to study the role of key residues in the binding pocket. Methods: We express mouse melanopsin which has a 1D4 epitope tag appended to its C–terminus. Expressed melanopsin is reconstituted with chromophore and then purified using immuno–affinity column chromatography. Purified melanopsin is analyzed using a spectrophotometer to determine the wavelength absorbance spectrum for the photopigment. Site directed mutagenesis was performed using QuikchangeTM. Results: We have recently observed that in the absence of salt the expressed melanopsin pigment has a dramatic red shift in its spectral sensitivity. In our mutagenesis study we mutated residue Y145 to a glutamic acid. In a clustal alignment with bovine rhodopsin, Y145 aligns with the rhodopsin counterion E113. Spectral analysis of the Y145E mutant shows that the addition of an acidic residue at this position causes the pigments spectral sensitivity to red shift. Conclusions: These results show that melanopsin forms a functional photopigment in vitro, and that the solubilized expressed melanopsin pigment is sensitive to the salinity of the assay environment. These results also support the hypothesis that melanopsin is involved in the regulation of photoentrainment of the circadian clock in mammals.
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