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M.T. Walker, R.L. Brown, T.H. Cronin, P.R. Robinson; Melanopsin in Solution Forms Two Spectrally Distinct Pigment Isoforms in the Dark . Invest. Ophthalmol. Vis. Sci. 2006;47(13):813.
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Melanopsin is a novel retinal G–protein coupled receptor (GPCR) which is expressed in a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) in the inner retina of mammals. The light–dependent response in ipRGCs has been demonstrated to be most sensitive to 480nm light, and melanopsin is also necessary for this response. In in vitro systems 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. These properties of melanopsin suggests that it may be the primary photopigment initiating the light response in ipRGCs, but unlike other vertebrate visual pigments, melanopsin’s absorbance maximum of 420nm when solubilized in detergent is drastically blue–shifted from the wavelength sensitivity of ipRGCs. Although this type of spectral difference between in vivo and in vitro data has not been observed in vertebrate visual pigments, it has been reported in some invertebrate visual pigments. In this investigation we demonstrate that melanopsin solubilized in detergent is sensitive to the ionic strength of the solution.
The expressed melanopsin has a 1D4 epitope tag (the last 15 amino acids of Bovine Rhodopsin) 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. Purified membranes containing melanopsin are used in a filter–binding assay to determine the photopigments rate of G–protein activation
Solubilized in a detergent solution with high or low NaCl concentration, melanopsin forms two spectrally distinct isoforms of the photopigment, with absorbance maxima of 420nm and 480nm respectively. We also show that heterologously expressed melanopin in the plasma membrane of COS cells activates Gt with an increased sensitivity to 480nm over 420nm.
These data show that the heterologously expressed melanopsin can form a photopigment that matches the in vivo wavelength sensitivity, and further suggests that melanopsin is the photopigment that initiates the photoresponse of ipRGCs. These results also suggest that along with increased amino acid sequence similarity to invertebrate opsins, melanopsin may also share some biochemical similarities.
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