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Shinya Sato, Beata Jastrzebska, Andreas Engel, Krzysztof Palczewski, Vladimir J Kefalov; Apo-opsin forms a photoactivated rhodopsin-like state. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5602.
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© ARVO (1962-2015); The Authors (2016-present)
Bleaching adaptation in rods is mediated by apo-opsin, which activates phototransduction with an estimated activity 106-fold lower than that of photoactivated rhodopsin (Meta-II). It is unclear whether every opsin molecule has low constitutive activity or if opsin exists in equilibrium between a predominantly inactive state and an intermittent active state. To address this question, we studied opsin signaling by electrophysiological recordings from mouse rods.
We studied opsin signaling in two models, guanylate cyclase activating proteins knockout mice (GCAPs-/-) and retinal pigment epithelium specific 65 kDa protein knockout mice (RPE65-/-). First, we examined GCAPs-/- mouse rods, which have ~5 times higher sensitivity than wildtype rods, in an effort to resolve the signal from individual opsins. Prior to the recordings, dark-adapted mouse retinas were dissected and a small fraction of opsin was produced by bleaching <1% of rhodopsin by light. Then, activation of the phototransduction cascade by opsin was measured from rod outer segments by single-cell suction recordings in the dark. Next, we stueied RPE65-/- chromophore-deficient rods. Here, prior to recordings, almost all of the opsin was converted into unbleachable rhodopsin by regeneration with exogenous locked 11-cis-7-ring retinal. Resistance of this 11-cis-7-ring rhodopsin to photoactivation and bleaching was confirmed biochemically. The signaling of the residual small fraction of apo-opsin in these rods was then measured by the same methods as above.
Surprisingly, we observed frequent photoresponse-like events in the dark from bleached GCAPs-/- rods. The rate of these photoresponse-like events was similar from 2 hours to 12 hours after the bleach, arguing against a contribution from Meta-II. Consistent with this interpretation, dark activity returned to pre-bleached levels by regenerating bleached opsin into rhodopsin with exogenous 11-cis retinal treatment. These data suggest that opsin can form an active Meta-II like state. We observed similar events in RPE65-/- rods regenerated with the unbleachable rhodopsin analogue, further ruling out the involvement of Meta-II and its decay intermediates in these photoresponse-like events.
Our data suggest that, contrary to current beliefs, bleaching adaptation in rods is mediated by opsin that exists in equilibrium between predominantly inactive and intermittently Meta-II like states.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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