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Tomoki Isayama, Junchao Wu, Sadaharu Miyazono, Vanessa Lee, Emily Levine, Elina Makino, Anita Zimmerman, Clint Makino; Modulating the phototransduction cascade with small molecules. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4076.
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© ARVO (1962-2015); The Authors (2016-present)
The weak constitutive activity of apo-opsin is quenched when the inverse agonist 11-cis retinal binds at its orthosteric binding site. Subsequent photoisomerization of 11-cis retinal to the all-trans conformation transforms the inverse agonist into an agonist that fully activates opsin. Certain retinal analogs can activate opsin in darkness. Some can even activate opsin with 11-cis retinal bound covalently, suggesting that they operate allosterically. Retinal analogs also inhibit the cyclic nucleotide-gated (CNG) cation channel of photoreceptors. The purpose of this study was to identify non-retinoid, allosteric ligands for opsin and the channel.
A screen of bovine opsin using homogeneous time resolved fluorescence identified a short list of possible agonists and inverse agonists for opsin. The effects of these compounds on the phototransduction machinery of intact, dark-adapted and partially bleached salamander rods and cones were assessed by suction electrode recording. Patch clamp recordings of membranes excised from salamander rod outer segments were carried out to test for inhibition of the CNG channel.
The cathepsin inhibitor CA-074ME, acted as an inverse agonist to opsin in partially bleached rods, supporting recovery of sensitivity and response kinetics in the presence of the ligand, but interestingly, response amplitude was unchanged. Membrane patches excised from salamander rod outer segments revealed that CA-074ME inhibited current through CNG channels, explaining the absence of the expected increase in circulating current. These effects, whether on the pigment or on the channel, were reversed when the compound was washed away. Another compound, D-pyroglutamic acid acted as an allosteric opsin agonist when applied to dark-adapted red-sensitive cones; there was a decrease in response amplitude, a loss in sensitivity to flashes, and accelerated response kinetics. Washing out D-pyroglutamic acid, however, did not reverse the effects, suggesting that visual pigment bleaching had occurred.
The catalytic activity of salamander visual pigment/opsin can be modulated by small molecules, at least some of which act allosterically. Some compounds also inhibited the CNG channels, suggesting that opsins and the channel may share one or more allosteric binding sites. These small molecules may prove to be useful in developing a therapy for certain types of inherited retinal diseases.
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