Abstract
Purpose :
Point mutations of Glycine 90 to Aspartate (G90D) or to Valine (G90V) in rhodopsin cause human congenital stationary night blindness (CSNB) and retinitis pigmentosa (RP), respectively. In vitro studies have shown that the G90D/G90V mutations decrease the stability of both chromophore-bound rhodopsin and chromophore-free opsin. This lower stability could produce persistent activation of rod photoreceptors even in darkness and interfere with normal detection of light, causing CSNB or RP. Yet, the molecular mechanism by which these mutations cause abnormal photoreceptor function remains controversial.
Methods :
We previously created homozygous mutant mice expressing either G90D or G90V rhodopsin. Here, we quantified rhodopsin and opsin content in whole mouse eyes and measured the chemical and thermal stabilities of purified pigments, the decay of their photoproducts, and their regenerability with exogenous 11-cis-retinal. We also followed the decay of Meta III in intact mouse rods by microspectrophotometry (MSP) on perfused retinas. The rod flash sensitivity was assessed by electroretinography ex vivo.
Results :
Unlike WT rhodopsin, both purified G90D and G90V mutants were chemically bleached by 50 mM hydroxylamine in the dark. However, only G90V mutant was thermally unstable at 48 oC. Both mutant lines had ~2 times lower dark rhodopsin content in their eyes compared to that in WT. While the level of chromophore-free opsin was low in dark-adapted WT and G90D retinas, G90V retinas contained a substantial fraction of free opsin. Consistent with that, treatment of dark-adapted retinas with 11-cis-retinal improved rod flash sensitivity in G90V but not G90D or WT mice. Biochemical studies revealed that the decay of Meta II was ~2 times faster (G90D) or ~2 times slower (G90V) compared to that of purified WT pigment. MSP recordings found that, in contrast to WT rhodopsin, the extensive conversion of Meta II to Meta III after a full bleach was not observed in G90 mutant retinas. Finally, both mutants (especially G90V) had reduced ability to regenerate with 11-cis-retinal in vitro.
Conclusions :
While both G90D and G90V mutations in rhodopsin compromise pigment stability, only the G90V mutant desensitized rods due to the presence of a fraction of free opsin in the dark. The mechanisms by which these two mutations cause different forms of visual impairment are currently being investigated.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.