The Y99C Mutation in Guanylyl Cyclase-Activating Protein 1 Increases Intracellular Ca²⁺and Causes Photoreceptor Degeneration in Transgenic Mice

Abstract

Guanylyl cyclase-activating proteins (GCAPs) are Ca 2ϩ -binding proteins that activate guanylyl cyclase when free Ca 2ϩ concentrations in retinal rods and cones fall after illumination and inhibit the cyclase when free Ca 2ϩ reaches its resting level in the dark. Several forms of retinal dystrophy are caused by mutations in GUCA1A, the gene coding for GCAP1. To investigate the cellular mechanisms affected by the diseased state, we created transgenic mice that express GCAP1 with a Tyr99Cys substitution (Y99C GCAP1) found in human patients with a late-onset retinal dystrophy (Payne et al., 1998). Y99C GCAP1 shifted the Ca 2ϩ sensitivity of the guanylyl cyclase in photoreceptors, keeping it partially active at 250 nM free Ca 2ϩ , the normal resting Ca 2ϩ concentration in darkness. The enhanced activity of the cyclase in the dark increased cyclic nucleotide-gated channel activity and elevated the rod outer segment Ca 2ϩ concentration in darkness, measured by using fluo-5F and laser spot microscopy. In different lines of transgenic mice the magnitude of this effect rose with the Y99C GCAP1 expression. Surprisingly, there was little change in the rod photoresponse, indicating that dynamic Ca 2ϩ -dependent regulation of cGMP synthesis was preserved. However, the photoreceptors in these mice degenerated, and the rate of the cell loss increased with the level of the transgene expression, unlike in transgenic mice that overexpressed normal GCAP1. These results provide the first direct evidence that a mutation linked to congenital blindness increases Ca 2ϩ in the outer segment, which may trigger the apoptotic process.

Evaluating the Role of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Domains in Binding Guanylyl Cyclase-activating Proteins (GCAPs)

“…Triple RetGC2 Ϫ/Ϫ GCAPs1,2 Ϫ/Ϫ (11) gene knock-out mice lack RetGC2 isozyme and both GCAPs altogether, but RetGC1 in these retinas is expressed at a normal ratio to rhodopsin and preserves its activation and normal Ca 2ϩ -sensitive regulation in the presence of exogenously added GCAP1 and GCAP2 (11). Note that this cyclase assay in whole retina specifically measures activity of RetGC from photoreceptors (11,33,34). We found that the native RetGC1 in the triple knock-out retinas saturated by GCAP1 did not increase its activity when GCAP2 was also added in the same assay.…”

Why photoreceptors die (and why they don't)

“…Although it remains unclear why excess stimulation would do this, our best guess at present is that continuous activation triggers apoptosis by producing a prolonged lowering of the Ca 2þ concentration. Since high Ca 2þ can also kill photoreceptors, (49) these cells appear to tolerate a Ca 2þ concentration only within a restricted range, much as has been proposed for neurons. (91) Future experiments with animal models having altered cyclic nucleotidegated channels and transporters may help define this range with more precision.…”

“…48), and high outer segment Ca 2þ has recently been indicated as a probable precipitator of apoptosis in some kinds of retinal degenerations. (49) For Ca 2þ to be the agent of death during continuous light and vitamin A deprivation, however, apoptosis would have to be triggered not by an increase in Ca 2þ but rather by a maintained decrease. In real or equivalent background light, the probability of opening of a cGMP-gated channel decreases (Fig.…”

The R838S Mutation in Retinal Guanylyl Cyclase 1 (RetGC1) Alters Calcium Sensitivity of cGMP Synthesis in the Retina and Causes Blindness in Transgenic Mice

“…Because the mutant cyclase binds the activator form of GCAP1 with higher affinity (44 -46), it now takes higher Ca 2ϩ concentrations, converting a larger fraction of GCAP1 into the inhibitor state, to outcompete the activator form of GCAP1 and decelerate the cyclase (46). Deregulation of RetGC1 is the main reason for photoreceptor death in mouse models expressing Y99C or E155G GCAP1 mutants (58,62,63). When RetGC1 deceleration by Ca 2ϩ in the dark becomes less sensitive, a larger fraction of cGMP-gated channels in the plasma membrane maintains Ca 2ϩ influx in the dark, eventually provoking the onset of apoptosis (62,63).…”

“…Deregulation of RetGC1 is the main reason for photoreceptor death in mouse models expressing Y99C or E155G GCAP1 mutants (58,62,63). When RetGC1 deceleration by Ca 2ϩ in the dark becomes less sensitive, a larger fraction of cGMP-gated channels in the plasma membrane maintains Ca 2ϩ influx in the dark, eventually provoking the onset of apoptosis (62,63). Stimulation of cGMP-dependent protein kinase activity can also lead to mouse photoreceptor death independently of the Ca 2ϩ pathway (64,65).…”