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W Baehr, ME Pennesi, I Sokal, D Margolis, JM Frederick, F Rieke, K Palczewski, SM Wu, PB Detwiler, KA Howes; GCAP1 Rescues Rod Photoreceptor Response in GCAP1/GCAP2 Knockout Mice . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1416.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In retinal photoreceptors, Ca2+ regulates the activity of guanylate cyclase (GC) and the synthesis of cGMP through a GC-activating protein (GCAP). The GCAP responsible for this process has not been identified. We tested if GCAP1, one of two GCAPs present in mouse rods, supports the generation of normal flash responses. Methods: The entire mouse GCAP1 gene (consisting of 5.4 kb upstream regulatory region, all exons and introns and a polyadenylaytion site) was used to generate transgenic mice. Immunocytochemistry was performed with GCAP1 and recoverin monospecific antibodies. Retinas expressing GCAP1 transgene on a GCAP null background were analyzed by paired flash ERGs and single rod cell recordings. Results:Two stable transgenic lines expressing wild-type mouse GCAP1were obtained, G1T3 and G1T4. G1T3+ mice carry a deletion of 1.8 kb in the proximal promoter region, while G1T4+ mice carry an intact promoter. Crossbreeding of GCAP+/- and G1T+GCAP+/- mice generate phenotypes expressing GCAP1 (or no GCAP1) on GCAP+/+, GCAP+/-, and GCAP-/- backgrounds. G1T3+GCAP-/- mice were unable to express significant levels of GCAP1 in rods, but show enhanced expression of GCAP1 in cones. G1T4+GCAP-/- mice express GCAP1 in rods and cones, but expression in rods varies, with a majority of rods expressing GCAP1 at near normal levels. In paired flash ERGs (high bleaching levels), G1T4+GCAP-/- retinas showed recovery approaching approximately 70-80% of wild-type, while responses from G1T3+GCAP-/- retinas were much closer to GCAP-/- retinas. In single rod cell recordings (low bleaching levels), flash responses from a majority of dark-adapted G1T4+GCAP-/- rods were identical to responses from wild-type rods. Flash responses of rods from the G1T3+ line (G1T3+GCAP-/-) do not show a similar rescue of wild-type properties, but were more similar to the responses recorded from GCAP-/- rods. Single cell responses from rods of mice overexpressing GCAP1 (G1T4+GCAP+/+) and paired flash responses from G1T4+GCAP+/+ retinas closely resemble wild-type. Conclusion: Single cell recordings of G1T4+ rods on a null background demonstrate that GCAP1 at near normal levels supports the generation of wild-type flash responses in the absence of GCAP2.
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