Abstract
Purpose :
There is a total of 16, mostly hydrophobic, amino acid residues different between mouse and human rod visual pigments. It is not clear whether one or more of these distinct sites contribute to structural and functional differences between the two rhodopsins and the rate of rod dark adaptation in nocturnal (mouse) vs. diurnal (human) species. We characterized the biochemical and physiological properties of mouse rhodopsin carrying a point mutation of Phenylalanine-88 to Leucine (F88L), with the latter residue present in human pigment. That site is located in the second transmembrane domain and surrounded by a cluster of hydrophobic residues conserved between the two opsins. There are no known human blinding diseases associated with the residue.
Methods :
Using CRISPR/Cas9 technology, we created homozygous mutant mice with F88L rhodopsin and performed their initial characterization. Rhodopsin content, the decay of its photoproducts, chemical and thermal stabilities, and regeneration with 11-cis-retinal were measured by spectrophotometry of immunopurified pigments from mouse retinas. Retinal morphology was analyzed by hematoxylin and eosin staining of retinal cross-sections. Scotopic function and rod dark adaptation were assessed by in vivo electroretinography. Overall rod-mediated vision was evaluated using optomotor responses.
Results :
The expression level as well as the chemical stability of F88L rhodopsin in 50 mM hydroxylamine were normal in 3-month-old mutant mice. Surprisingly, F88L rhodopsin had increased thermal stability at 55 oC as compared to WT rhodopsin. The postbleach decay of mutant metarhodopsin II was accelerated at 37 oC as compared to that of WT pigment. The mutant also regenerated with 11-cis-retinal more efficiently in vitro. Retinal morphology and scotopic function were normal in F88L mice for up to 14 months of age, but their rod dark adaptation appeared slightly faster than in controls. Scotopic vision was not reduced in old F88L animals.
Conclusions :
Our results demonstrate that substituting a single amino acid at position 88 of rhodopsin from the mouse (Phe) to human (Leu) residue alters some molecular properties of the pigment. Distinct biochemical properties of mouse rhodopsin with a “humanized” F88L point mutation at a non disease-associated amino acid position may indicate evolutionary adjustment of rod opsins to better perform in specific light conditions.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.