The photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in phototransduction and cellular calcium homeostasis. Mutations in the cone photoreceptor CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. CNG channel deficiency leads to endoplasmic reticulum (ER) stress-associated cone apoptosis, protein mislocalization, and ER calcium dysregulation. This work investigated the potential mechanisms of protein mislocalization associated with ER calcium dysregulation using Cnga3(-/-) mice lacking ER Ca2+ channel ryanodine receptor 2 (RyR2) specifically in cones. Deletion of Ryr2 improved outer segment (OS) localization of the cone proteins M-opsin, S-opsin, and cone phosphodiesterase subunit alpha' (PDE6C) and decreased inner segment localization. One-month-old Cnga3(-/-) mice showed similar to 30% of M-opsin, 55% of S-opsin, and 50% of PDE6C localized to the OS. Cnga3(-/-) mice with Ryr2 deletion at the same age showed almost 60% of M-opsin, 70% of S-opsin, and 70% of PDE6C localized to the OS. Deletion of Ryr2 nearly completely reversed elevations of the ER stress markers phospho-IRE1 alpha and phospho-eIF2 alpha and suppressed cone apoptosis. Consistent with the improved cone protein localization and reduced ER stress/cone apoptosis, cone survival was improved by deletion of Ryr2. The number of cones was increased by similar to 28% in 2- to 4-month-old Cnga3(-/-) mice with Ryr2 deletion compared with age-matched Cnga3(-/-) mice. This work demonstrates a role of RyR2/ER calcium dysregulation in protein mislocalization, ER stress, and cone death. The findings provide novel insights into the mechanisms of photoreceptor degeneration and support strategies targeting ER calcium regulation to manage retinal degeneration.

A new method for catalyst selection-optimization is introduced and evaluated, transition state affinity chromatography (TSAC), based on the relative chromatographic affinity of pre-catalysts for a supported substrate vs. a transition state analog (TSA). The affinities of a library of Zn-imine complexes on three designer HPLC affinity columns that possess either an immobilized substrate, a transition state analog or a non-binding reference compound are compared to their catalytic activities for picolinate ester hydrolysis. For those Zn-complexes whose ligands possess a hydroxyalkyl side chain the retention times on the substrate-affinity column correlate linearly with the catalyst-substrate affinity, 1/K-M, derived from the kinetics of the LZn-catalyzed hydrolysis of 4-nitrophenyl picolinate. Additionally, the kinetically determined esterolytic catalytic activities, k(cat), for the hydroxyalkyl-bearing complexes also correlate with their relative chromatographic affinity on the TSA-vs Sub-affinity columns. Zinc-complexes that lack the hydroxyalkyl arm, however, show no correlation of their chromatographic behavior with 1/K-M and an inverse correlation with k(cat). These results are interpreted in terms of differences in the catalytic mechanisms for the two sets of catalysts. TSAC is shown to be viable for selecting the most active esterolytic Zn-catalyst from a mixture of five complexes. (C) 2017 Elsevier B.V. All rights reserved.