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A. M. Dizhoor, X.-H. Wen, E. V. Olshevskaya, I. V. Peshenko, C. L. Makino; Altered Guanylyl Cyclase (retGC) Regulation and Rod Flash Response Kinetics in Guanylyl Cyclase Activating Protein 2 (GCAP-2) Gene Knockout Mice. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1680.
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In vertebrates, two GCAP isoforms, GCAP-1 and GCAP-2, activate photoreceptor guanylyl cyclase (retGC) in a Ca(2+) sensitive manner. In vitro, GCAP-2 is more sensitive to Ca(2+) than GCAP-1. A controversy exists in the field regarding the involvement of GCAP-2 in the rod photoresponse [1, 2]. The purpose of this study was to evaluate the role of GCAP-2 in mouse rods using GUCA1B gene knockout.
We replaced the first exon together with the putative promoter region of murine GUCA1B gene by a PGK:Neo:PGKtts cassette. The knockout mice were analyzed for photoreceptor protein content, retGC activity and electrophysiological responses.
In the knockout mice, GCAP-2 was not detectable by immunoblotting nor was there GCAP-2 specific immunofluorescence in the retina, which is normally abundant in rod outer segments and their synaptic termini. There were no compensatory changes in the levels of retGC1, retGC2 and GCAP-1 expression in the knockout mice. RetGC regulation in the absence of GCAP-2 was altered in two ways: a) Ca(2+) sensitivity shifted to higher than in wild type free Ca(2+) concentrations and b) maximal retGC activity at low free Ca(2+) concentrations decreased by two-fold. As a result, activation of the cyclase was unchanged at free Ca(2+) above 100 nM, but fell below that of wild type as free Ca(2+) dropped to the concentrations typical of light-adapted rods. Dim flash responses of knockout rods recovered slower and bright flash responses remained in saturation longer than normal. Consequently, knockout rods were more sensitive to steps of light and saturated at lower intensities when compared to wild type.
GCAP-2 contributes to the full regulation of cyclase activity over the physiological range of Ca(2+) in rods in vivo. GCAP-1 operates at Ca(2+) concentrations closer to those present in dark adapted rods, while GCAP-2 takes over as Ca(2+) falls to the low concentrations characteristic of the fully light adapted state. GCAP-2 quickens the electrical photoresponse recovery after bright as well as after dim flashes. By limiting the integration time of the single photon response, GCAP-2 enables rods to withstand higher ambient lighting intensities before saturating.References:  Mendez et al., Proc Natl Acad Sci U S A. 14: 9948 (2001);  Howes et al., EMBO J. 21:1545 (2002).
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