Abstract:

The two key cycles that are essential to sustain vision are the photocycle of visual opsins and the retinoid or visual cycle. Visual opsins include rhodopsin and cone opsin, which are densely packed in the outer segment of rod and cone photoreceptor cells, respectively. The rhodopsin photocycle was first described by Böll and Kühne in the 1870s, inspiring many major breakthroughs in our understanding of visual physiology. Vision starts when a photon of light isomerizes the 11-cis-retinylidene chromophore bound in Schiff base linkage to rhodopsin. The resultant all-trans-retinylidene agonist induces a series of conformational changes in photoactivated rhodopsin, leading to the signaling state to elicit phototransduction. Schiff base hydrolysis of the agonist leads to the release of all-trans-retinal, which must be recycled into 11-cis-retinal by the visual cycle. Apo-opsin binds 11-cis-retinal to regenerate rhodopsin pigment for another round of light detection. As such hydrolysis serves as the key step diminishing ongoing phototransduction, as well as bridging both the visual cycle and the photocycle of visual opsins. 

Many challenges have been encountered in measuring the rate of this hydrolysis, which has largely been studied indirectly by optical spectroscopy and electrophysiology techniques. Our studies demonstrate the ability to directly measure hydrolysis using a method developed in-house, utilizing liquid chromatography-tandem mass spectroscopy (LC-MS/MS) and a specialized sample preparation involving sodium borohydride in isopropanol (NaBH4/iPrOH). NaBH4/iPrOH led to simultaneous protein denaturation and reductive trapping of chromophore as a retinyl moiety in secondary amine linkage to opsin. Concurrently, alcohol-induced protein precipitation facilitated fractionation of proteins from lipids to quantitate the amount of agonist remained bound to opsin, measured in the protein fraction, or hydrolyzed as all-trans-retinal, measured in the lipid fraction. Subsequent steps of N-retinylidene-phosphatidylethanolamine adduct formation and reduction to all-trans-retinol by retinol dehydrogenases were also detected by LC-MS/MS of the lipid fraction. Altogether each successive step of the visual cycle occurring within the rod outer segment was determined for its rate in native membranes, with each successive step an order of magnitude faster than the prior.

Our method was applied to other visual and non-visual opsins, namely cone opsins, retinal G protein-coupled receptor (RGR), and peropsin (RRH). Schiff base hydrolysis of agonist in photoactivated cone opsins was found to be markedly faster than in photoactivated rhodopsin. Reciprocally, RGR displayed trans-cis photochemistry and subsequent rapid hydrolytic release of 11-cis-retinal, followed by immediate re-binding of all-trans-retinal. The rapid rate of hydrolysis and regeneration was observed for RGR in microsomal membranes of the retinal pigment epithelium. As such, RGR plays a crucial role in continuously supplying chromophore needed to sustain photopic or color vision under bright daylight conditions. Though RRH was suspected to function similarly to RGR in the photic production of 11-cis-retinal from all-trans-retinal, RRH did not exhibit pigment formation with all-trans-retinal or any mono-cis retinal isomers, leaving uncertain its role in visual physiology.