Photoreceptor cell death following rise in intracellular free Ca2+ concentration in rods of transgenic mice expressing the Y99C mutant of guanylyl cyclase–activating protein 1 (GCAP1).

Abstract

Abstract: : Purpose. Mutations in GCAP1 have been found in patients with dominant cone and cone–rod dystrophies. Some of them, including Y99C, shift inhibition of guanylyl cyclase (retGC) by free Ca²⁺ in vitro to higher concentrations [1,2]. We explored physiological and biochemical changes in vivo caused by the Y99C GCAP1 in photoreceptors of transgenic mice. Methods. Wild type bGCAP1 and its Y99C mutant were expressed under control of a cone/rod–specific promoter in transgenic mice, and photoreceptor function was evaluated using electroretinography (ERG) and single–rod recording along with morphological analysis of the retinas at different ages. The free Ca²⁺ concentration in the outer segment of the Y99C mouse rods was determined using laser spot microscopy and a fluorescent indicator dye [3]. Results. In the Y99C mice the dynamic range for retGC regulation by Ca²⁺ was shifted to higher submicromolar concentrations than in normal siblings or in mice overexpressing normal GCAP1. By six months of age the Y99C mice dramatically reduced or completely lost their ERG responses. The loss of rod and cone function was accompanied by photoreceptor death, and the rate of degeneration scaled with the level of the Y99C GCAP1 expression. No functional or morphological evidence for retinal dystrophy was observed in mice that overexpressed normal GCAP1. Interestingly, the Y99C GCAP1 did not dramatically alter flash response kinetics or light sensitivity in rods, but the free Ca²⁺ in the dark was increased, in some lines by almost two–fold, and the extent of Ca²⁺ increase correlated with the rate of degeneration. Free Ca²⁺ in Y99C mice in the light decreased to the same level as in non–transgenic siblings. Conclusions. Unlike overexpression of normal GCAP1, Y99C GCAP1 causes photoreceptor death. The shift in Ca²⁺ sensitivity of RetGC regulation by the Y99C GCAP1 can increase RetGC activity in the dark, and thus produce a higher probability of the cGMP gated channels opening. This may account for the increase in free Ca²⁺ found in the Y99C mouse rods. Free Ca²⁺ rise caused by the Y99C GCAP1 can contribute to, or even itself be the cause of apoptosis in photoreceptors. References. [1] Dizhoor et al.,1998, J Biol Chem. 273, 17311;[2] Wilkie et al., 2001, Am J Hum Genet. 69, 471; [3] Woodruff et al., 2002, J.Physiol. 542,843.

Keywords: retinal degenerations: cell biology • transgenics/knock–outs • photoreceptors