Regulation by Ca^ binding proteins

In photoreceptors, Ca^^ play a crucial role in photorecovery and adaptation (Matthews et al., 1988). Calcium ions regulate several stages of the phototransduction pathway by modifying the activity o f different Ca^ binding proteins that in turn interact with key enzymes in the pathway. In the dark, the concentration of Ca^ is maintained at 300nM as the entry o f Ca^^ through the cGMP-gated cation channels is balanced by efflux o f calcium through the Na7Ca^^-K^ exchanger (Koch and Stryer, 1988). Upon light illumination, the closure of cGMP-gated cation channels and the continued efflux of calcium through the Na^/Ca^^-K^ exchanger result in the drop of Ca^ concentration to <70 nM. This decrease in [Ca^^] stimulates the enzyme guanylate cyclase (GC), the peripherally membrane bound enzyme that catalyses the conversion of GTP to cGMP. Following illumination, this key enzyme is responsible for the synthesis o f cGMP, which in turn opens cation channels in the outer segment plasma membrane and re-establishes the dark potential o f the cell. Two retina-specific membrane-associated guanylate cyclases have been cloned and sequenced (retGCl and retGC2) (Shyjan et al., 1992; Margulis et al., 1993; Lowe et al., 1995), however only retGCl has been localised to outer segments by immunocytochemical localisations (Dizhoor et al., 1994; Liu et al., 1994). These GCs are activated and regulated by specific Ca^^-binding proteins known as guanylate cyclase activating proteins (GCAPs) (Gorczyca et a l, 1995; Dizhoor et a l, 1995; Palczewski et a l, 1994). Two GCAP proteins have been isolated from retina (GCAPl and GCAP2), but only GCAPl have been localised definitively to both rod and cone outer segments and purified. Ca^^ free form o f GCAPl has been shown to regulate the activity o f ROS guanylate cyclase as well as recombinant retGCl (Subbaraya et a l.

1994; Gorczyca et al., 1995). Therefore m summary GCAPs mediate Ca^^ sensitive regulation o f guanylate cyclase, which by synthesising cGMP restores the open state of the channels, thus promoting recovery of the dark state of rod photoreceptors following light exposure. The entry of Ca^^ then leads to decreased activity of GC and the return of the dark state. This feed back loop involving Ca^^ is likely to be a major contributor in the maintenance of a constant cGMP level in the dark and to recovery following illumination (Koch and Stryer, 1988).

Originally a different photoreceptor-specific Ca^^-binding protein, recoverin, was thought to be the regulator of rod outer segment guanylate cyclase activity (Dizhoor et a l, 1991; Lambrecht and Koch, 1991), but once cloned and expressed recoverin did not alter the activity of GC under in vitro conditions (Hurley et al., 1993; Gray-Keller et al., 1993). It was also demonstrated that raising the concentration of recoverin within rod cells slows recovery from photoexcitation. Moreover, Visinin and S-modulin (Gray- Keller et al., 1993) the recoverin like proteins isolated from chicken cones and frog rods, respectively, were also shown to prolong the activation of cGMP phosphodiesterase (PDE) (Kawamura and Murakami, 1991). Since the proposed function for recoverin in the regulation of GC was not supported, the precise role of recoverin in the phototransduction pathway has been intensely investigated. Recent studies o f rhodopsin phosphorylation has revealed a possible function for recoverin and related proteins. S- modulin was shown to inhibit phosphorylation at elevated levels o f Ca^^ (Kawamura, 1993; Klenchin et al., 1995). Because phosphorylation of rhodopsin and subsequent binding o f arrestin block further activation of transducin, thus reducing the effective lifetime o f photolysed rhodopsin, the inhibition of phosphorylation by S-modulin and recoverin would be expected to prolong the lifetime o f activated rhodopsin. This is also an effect consistent with the longer lifetime of activated PDE and the prolonged photoresponse. Recoverin is now thought to function during light adaptation through its Ca^^-dependent inhibition of rhodopsin kinase (Klenchin et al., 1995; Gorodovikova and Philippov, 1993; Gorodovikova et al., 1994), an idea further supported by the observation that recoverin binds to rhodopsin kinase in a Ca^^-dependent manner (Chen et al., 1995a). However the transgenic mouse in which recoverin expression has been eliminated does not show the expected response kinetics (Baylor, 1996). In addition there

is disagreement about the Ca^^ concentration at which recoverin might regulate the phosphorylation of rhodopsin, perhaps requiring a concentration elevated beyond physiological conditions. Furthermore recoverin immunoreactivity is most prevalent at photoreceptor terminals (Polans et al., 1993; Milam et al., 1993), and unlike other phototransduction-specific proteins which are sequestered in the outer segments, recoverin is distributed throughout the cell, indicating that it might be involved in functions distinct from phototransduction. Therefore the precise function of recoverin in phototransduction is still controversial. Aside from studies o f phototransduction, recoverin was also identified as the protein previously known as CAR (cancer associated retinopathy) protein. The gene for the mouse recoverin protein was originally assigned to mouse chromosome 11, closely linked to trp53. In the paper, the human gene for recoverin was localized to human chromosome 17 by Southern analysis o f restriction digests of the DNA from mouse/human somatic cell hybrids. Using a 7 kb subclone of the human recoverin gene, a positive fluorescence in situ hybridization signal was demonstrated near the terminus of the short arm of chromosome 17 at position pl3.1. The mapping o f recoverin to this region of human chromosome 17, which contained a number of cancer-related loci, suggested a possible mechanism by which cancer- associated retinopathy could occur in humans (McGinnis et al., 1995).

Calmodulin is another cytosolic Ca^-binding protein that is expressed ubiquitously and found in the rod outer segments (Nagao et al., 1987). It has been suggested that the cGMP-gated channels are also responsive to concentrations of Ca^^ and might be regulated by Calmodulin (Hsu and Molday, 1993). In vitro experiments have shown that in the dark, binding of Ca^^ bound calmodulin to the P-subunit of the channel protein lowers the apparent affinity of the channel for cGMP. In this low affinity state any decrease in cytosolic cGMP, as which occurs upon light illumination will lead to channel closure. The resultant lowering of intracellular Ca^^ levels due to channel closure will in turn increase the sensitivity o f the channel to cGMP by uncoupling calmodulin from the channel, thus allowing the channel to reopen at lower cGMP levels, leading to recovery of the ROS to its dark state as cGMP synthesis proceeds with the activity o f guanylate cyclase. The opening of the channel will in turn restore the [Ca^^] to dark levels and Ca^^ bound calmodulin will rebind the channel. All these have been

postulated upon in vitro experiments, however a Ca^^ binding protein other than calmodulin might contribute to channel sensitivity in vivo (Downing and Zimmerman,

1995).

Therefore in summary, the lowered Ca^ levels due to photon absorption; (1) accelerate the synthesis of cGMP owing to GC stimulation by the Ca^^-free form of GCAPl; (2) increase sensitivity of the channel to cGMP and accelerate the recovery of the dark current and Ca^ by uncoupling calmodulin from the cGMP gated channel; and (3) shorten the life time of photolysed rhodopsin (R*) by uncoupling recoverin from rhodopsin kinase, thus allowing the inhibitory effect of rhodopsin phosphorylation to proceed as described in the following section. These Ca^^ sensitive steps represent the principle mechanisms of light adaptation in vertebrate photoreceptors.