Chromophore hydrolysis and release from photoactivated rhodopsin in native membranes

Abstract

For sustained vision, photoactivated rhodopsin (Rho*) must undergo hydrolysis and release of all- trans -retinal, producing substrate for the visual cycle and apo-opsin available for regeneration with 11- cis -retinal. The kinetics of this hydrolysis has yet to be described for rhodopsin in its native membrane environment. We developed a method consisting of simultaneous denaturation and chromophore trapping by isopropanol/borohydride, followed by exhaustive protein digestion, complete extraction, and liquid chromatography–mass spectrometry. Using our method, we tracked Rho* hydrolysis, the subsequent formation of N -retinylidene-phosphatidylethanolamine ( N -ret-PE) adducts with the released all- trans -retinal, and the reduction of all- trans -retinal to all- trans -retinol. We found that hydrolysis occurred faster in native membranes than in detergent micelles typically used to study membrane proteins. The activation energy of the hydrolysis in native membranes was determined to be 17.7 ± 2.4 kcal/mol. Our data support the interpretation that metarhodopsin II, the signaling state of rhodopsin, is the primary species undergoing hydrolysis and release of its all- trans -retinal. In the absence of NADPH, free all- trans -retinal reacts with phosphatidylethanolamine (PE), forming a substantial amount of N -ret-PE (∼40% of total all- trans -retinal at physiological pH), at a rate that is an order of magnitude faster than Rho* hydrolysis. However, N -ret-PE formation was highly attenuated by NADPH-dependent reduction of all- trans -retinal to all- trans -retinol. Neither N -ret-PE formation nor all- trans -retinal reduction affected the rate of hydrolysis of Rho*. Our study provides a comprehensive picture of the hydrolysis of Rho* and the release of all- trans -retinal and its reentry into the visual cycle, a process in which alteration can lead to severe retinopathies.

Retinal-phospholipid Schiff-base conjugates and their interaction with ABCA4, the ABC transporter associated with Stargardt disease

“…During preparation of this article for submission, Hong et al . ( 44 ), using a different approach, published a paper showing that as much as 40% of the free retinal added to ROS formed N -Ret-PE, in broad agreement with our studies.…”

A short story on how chromophore is hydrolyzed from rhodopsin for recycling

“…[58] MII, the signaling state of Rho*, exists as the dominant species under physiological pH and has been shown to be the species from which the hydrolysis of the all-trans-retinylidene Schiff base largely proceeds (Figure 4). [26,27,61,62]…”

Profile of Krzysztof Palczewski

“…In his Inaugural Article ( 5 ), Palczewski provides an overview of his recent studies on the chemistry of vision. “I think there is still a lot of mystery in how our visual system operates, and there are still a lot of chemical questions that need to be answered,” he says.…”

“…For his Inaugural Article ( 5 ), Palczewski focused on a previously little-explored but critical step in vision. “When the absorption of light happens, the chromophore has to be released and, then, a fresh, newly synthesized active chromophore needs to replace it to make the retina light-sensitive again and to enable visual transduction,” he says.…”

“…Now director of the Center for Translational Vision Research and a professor of ophthalmology at the University of California, Irvine, Palczewski continues to work on deciphering the fundamental biochemical processes operating in the visual system. Palczewski, a member of both the National Academy of Sciences and the National Academy of Medicine, describes some of his latest discoveries in his Inaugural Article ( 5 ).…”