Abstract
Purpose :
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.
Methods :
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.
Results :
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.
Conclusions :
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.