Measurement of Slow Spontaneous Release of 11-cis-Retinal from Rhodopsin

Abstract

The vertebrate visual photoreceptor rhodopsin (Rho) is a unique G protein-coupled receptor as it utilizes a covalently tethered inverse agonist (11-cis-retinal) as the native ligand. Previously, electrophysiological studies showed that ligand binding of 11-cis-retinal in dark-adapted Rho was essentially irreversible with a half-life estimated to be 420 years, until after thermal isomerization to all-trans-retinal, which then slowly dissociates. This long lifetime of 11-cis-retinal binding was considered to be physiologically important for minimizing background signal (dark noise) of the visual system. However, in vitro biochemical studies on the thermal stability of Rho showed that Rho decays with a half-life on the order of days. In this study, we resolve the discrepancy by measuring the chromophore exchange rate of the bound 11-cis-retinal chromophore with free 9-cis-retinal from Rho in an in vitro phospholipid/detergent bicelle system. We conclude that the thermal decay of Rho primarily proceeds through spontaneous breaking of the covalent linkage between opsin and 11-cis-retinal, which was overlooked in the electrophysiological recording. We estimate that this slow spontaneous release of 11-cis-retinal from Rho should result in 10 4 to 10 5 free opsin molecules in a dark-adapted rod cell-a number that is three orders of magnitude higher than previously expected. We also discuss the physiological implications of these findings on the basal activity of opsins and the associated dark noise in the visual system.

Elementary response triggered by transducin in retinal rods

“…Incidentally, incubating unbleached, dark-adapted mouse rods with 11-cis-retinal did not have any obvious effect on dark noise (SI Appendix,Fig. S2), confirming previous reports of relatively negligible Opn in the absence of bleaching(28)(29)(30)(31).…”

The Energetics of Chromophore Binding in the Visual Photoreceptor Rhodopsin

“…Bicelles are model membranes that consist of long-chain phospholipids, which form the planar lipid bilayer segment, and detergents or short-chain phospholipids, which stabilize the edge of the bilayer. In an earlier report (12), we showed that dark-state Rho was stable in 1% (w/v) POPC/CHAPS bicelles over the timescale of weeks. Here, we evaluated the stability of opsin in POPC/CHAPS bicelles (Fig.…”

“…The first assay based on the intrinsic tryptophan (Trp) fluorescence of Rho has been described in Tian et al (12) and Farrens and Khorana (13). In brief, Trp fluorescence can be quenched by 11CR in the dark-state Rho (Fig.…”

“…2 F) showed that detergent micelles are ineffective for stabilizing ligand-free opsin. By comparison, a bicelle system better mimics the native membrane environment and effectively stabilizes free opsin at its functional state (12,22,23). Compared with liposomes (24), bicelles are easier to prepare, show much less light scattering, and are more compatible with UV-Vis and fluorescence spectroscopy.…”

“…We then measure the binding kinetics at several temperatures. In an earlier report (12), we have obtained the temperature-dependent kinetics for its reverse reaction, i.e., the light-independent, spontaneous dissociation rate of 11CR from Rho. These kinetics data enable us to calculate the activation enthalpy and entropic terms from the Eyring plot.…”