The Contribution of Endoplasmic Reticulum Calcium Deficiency to Mistrafficking of Cone Outer Segment Proteins in CNG Channel Deficiency

Abstract

Purpose : The photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in phototransduction and cellular calcium homeostasis. Mutations in cone CNG channel are associated with achromatopsia and cone dystrophies. We previously showed that CNG channel deficiency leads to endoplasmic reticulum (ER) stress-associated cone death and cone opsin mistrafficking. We also demonstrated enhanced expression/activity of the ER calcium releasing channels inositol 1,4,5-trisphosphate receptor 1 (IP₃R1) and ryanodine receptor 2 (RyR2) in CNG channel-deficient mice. This work investigated the potential mechanism of cone protein mistrafficking associated with ER calcium regulation in CNG channel deficiency.

Methods : Cnga3^-/- mice with cone-specific deletions of Ryr2 (encoding RyR2) or Itpr1 (encoding IP₃R1) were generated by cross mating. Cellular localization of cone outer segment proteins M-opsin, S-opsin and phosphodiesterase 6C (PDE6C) was examined by immunofluorescence labeling, confocal microscopy and ImageJ analysis. Cone survival was evaluated by peanut agglutinin (PNA) labeling. Expression of the ER chaperone proteins was evaluated by immunoblotting.

Results : Deletion of Ryr2 or Itpr1 improved outer segment (OS) localization of cone proteins in Cnga3^-/- mice. One-month-old Cnga3^-/- mice showed 27% of M-opsin, 56% of S-opsin, and 50% of PDE6C localized to the OS. Cnga3^-/-/Ryr2^fl/fl/Hrgp^Cre mice at the same age showed about 59% of M-opsin, 71% of S-opsin, and 70% of PDE6C localized to the OS, and Cnga3^-/-/Itpr1^fl/fl/Hrgp^Cre mice showed about 78% of S-opsin and 67% of PDE6C localized to the OS. Consistent with improved cone protein trafficking, cone density also significantly increased in Cnga3^-/- mice with deletion of Itpr1 or Ryr2. PNA positive cells increased by approximately 28% in ventral retinas of 2-month-old Cnga3^-/-/Ryr2^fl/fl/Hrgp^Cre mice, compared with Cnga3^-/- mice. Expression of the ER chaperone proteins Grp78/BiP and calreticulin was not affected by deletion of the ER calcium channels.

Conclusions : Deletion of Ryr2 or Itpr1 improves outer segment localization of cone proteins in CNG deficiency, suggesting a contribution of ER calcium deficiency to mistrafficking of cone outer segment proteins. This regulation appears to be mediated by an ER chaperone-independent mechanism.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.