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Fan Yang, Hongwei Ma, Michael Butler, Wolfgang Baehr, Martin Biel, Stylianos Michalakis, Xi-Qin Ding; cGMP/PKG Signaling Regulation of Endoplasmic Reticulum Calcium Channels in Cyclic Nucleotide-gated Channel-deficient Cones. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4966.
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Cone cyclic nucleotide-gated (CNG) channel deficiency leads to endoplasmic reticulum (ER) stress-associated cone degeneration. We have shown elevated cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) signaling in CNG channel deficiency and cone protection following cGMP depletion or PKG inhibition. We also found increased ER calcium channel activity in CNG channel deficiency contributing to ER stress and cone death. This work investigated the cGMP/PKG signaling regulation of ER calcium channels to understand how it induces ER stress and cone death.
Cnga3-/-/Nrl-/-/Gucy2e-/- mice which lack a CNG channel and retinal guanylate cyclase-1 (RetGC1) on a cone-dominant retina, along with Cnga3-/-/Nrl-/- and Nrl-/- mice, were used. The retinal expression levels and activity of ryanodine receptor 2 (RyR2) and 1,4,5-inositol triphosphate receptor 1 (IP3R1) and ER stress markers were evaluated by qRT-PCR and western blotting. The effects of cGMP/PKG signaling on ER calcium channels were also examined in the photoreceptor-derived WERI-Rb1 cells. Immunoprecipitation was performed to examine the interaction between PKG and ER calcium channels using Nrl-/- retinas.
Expression levels of IP3R1 and RyR2 were doubled in Cnga3-/-/Nrl-/- mice, and decreased by 50% in Cnga3-/-/Nrl-/-/Gucy2e-/- mice. The phospho-RyR2 level was doubled in Cnga3-/-/Nrl-/- and returned to control level in Cnga3-/-/Nrl-/-/Gucy2e-/- mice, whereas phospho-IP3R1 level was increased by 25% in Cnga3-/-/Nrl-/- and further increased by 20% in Cnga3-/-/Nrl-/-/Gucy2e-/- mice. PCR array and western blotting results showed the expression levels of 14 unfolded protein responsive (UPR) and ER stress genes were significantly increased in Cnga3-/-/Nrl-/- and reduced in Cnga3-/-/Nrl-/-/Gucy2e-/- mice. Treatment with cGMP significantly increased expression of IP3R1 and RyR2 in WERI-Rb1 cells. Immunoprecipitation revealed interaction of PKG1 with IP3R1 through inositol trisphosphate receptor-associated cGMP-kinase substrate (IRAG).
Our results showed elevated cGMP/PKG signaling induced the expression and activity of IP3R1 and RyR2, which may contribute to the dysregulation of ER calcium homeostasis and subsequent UPR and ER stress in CNG channel deficiency.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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