The death of photoreceptor cells caused by retinal degenerative diseases often results in a complete loss of retinal responses to light. We explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for imparting light sensitivity to retinas lacking rods and cones. Using delivery by an adeno-associated viral vector, here, we show that long-term expression of a microbial-type rhodopsin, channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinal neurons in vivo. Furthermore, we demonstrate that expression of ChR2 in surviving inner retinal neurons of a mouse with photoreceptor degeneration can restore the ability of the retina to encode light signals and transmit the light signals to the visual cortex. Thus, expression of microbial-type channelrhodopsins, such as ChR2, in surviving inner retinal neurons is a potential strategy for the restoration of vision after rod and cone degeneration.

Guanylate cyclase activating proteins, GCAP-1 and GCAP-2, have a pivotal role in the activation of guanylate cyclase in phototransduction. Previous studies on the localization of GCAP-1 and GCAP-2 are contradictory. In this study, we tried to avoid possible artifacts accompanied by immunocytochemistry. Immunolabeling of a GCAP was carried out using antibodies pre-adsorbed with a different type of GCAP. In addition, immunolabeling was performed using three different animal species under different fixation and embedding. Electron microscopic immunocytochemistry was also performed to reveal subcellular localization of GCAPs as well as confirming data obtained by light microscopy. All data indicate that anti-GCAP-1 antibody binding sites were found predominantly in cone outer segments, in particular, in disk membrane regions. Sparse labeling was observed in rod outer segments, but the labeling was much lower than that seen in cone outer segments. Less labeling is also found in synaptic regions and inner segments of cones. No labeling was detected in connecting cilia and its cytoplasmic extensions. Such labeling patterns were similar among human, monkey and bovine retinas. The localization of GCAP-1 is consistent with the pattern of a recently reported human cone-specific degeneration. Anti-GCAP-2 antibody binding sites were detected in both inner and outer segments of rods and cones of all three animals although the labeling density was slightly different among species. Cryo-immuno-labeling of GCAP-2 in bovine retinas revealed that labeling sites were more concentrated in rods than those of cones, and that synaptic regions were also labeled. The different localization of GCAPs suggest that roles of GCAP-1 and GCAP-2 may be different.

Retinal guanylyl cyclase-1 (retGC-1), a key enzyme in phototransduction, is activated by guanylyl cyclase-activating proteins (GCAPs) if [Ca2+] is less than 300 nM. The activation is believed to be essential for the recovery of photoreceptors to the dark state; however, the molecular mechanism of the activation is unknown. Here, we report that dimerization of retGC-1 is involved in its activation by GCAPs, The GC activity and the formation of a 210-kDa cross-linked product of retGC-1 were monitored in bovine rod outer segment homogenates, GCAPs-free bovine rod outer segment membranes and recombinant bovine retGC-1 expressed in COS-7 cells. In addition to recombinant bovine GCAPs, constitutively active mutants of GCAPs that activate retGC-1 in a [Ca2+]-independent manner and bovine brain S100b that activates retGC-1 in the presence of similar to 10 mu M [Ca2+] were used to investigate whether these activations take place through a similar mechanism, and whether [Ca2+] is directly involved in the dimerization. We found that a monomeric form of retGC-1 (similar to 110 kDa) was mainly observed whenever GC activity was at basal or low levels. However, the 210-kDa product was increased whenever the GC activity was stimulated by any Ca2+-binding proteins used. We also found that [Ca2+] did not directly regulate the formation of the 210-kDa product. The 210-kDa product was detected in a purified GC preparation and did not contain GCAPs even when the formation of the 210-kDa product was stimulated by GCAPs, These data strongly suggest that the 210-kDa cross-linked product is a homodimer of retGC-1. We conclude that inactive retGC-1 is predominantly a monomeric form, and that dimerization of retGC-1 may be an essential step for its activation by active forms of GCAPs.

Recoverin, a calcium ion (Ca2+)-binding protein of vertebrate photoreceptors, binds to photoreceptor membranes when the Ca2+ concentration is greater than 1 micromolar. This interaction requires a fatty acyl residue covalently linked to the recoverin amino (NH2)-terminus. Removal of the acyl residue, either by proteolytic cleavage of the NH2-terminus or by production of nonacylated recoverin, prevented recoverin from binding to membranes. The acylated recoverin NH2-terminus could be cleaved by trypsin only when Ca2+ was bound to recoverin. These results suggest that the hydrophobic NH2-terminus is constrained in Ca2+-free recoverin and liberated by Ca2+ binding. The hydrophobic acyl moiety of recoverin may interact with the membrane only when recoverin binds Ca2+.

Recoverin is a recently identified Ca2+-binding protein that imparts Ca2+ sensitivity to vertebrate photoreceptor guanylate cyclase. In response to photo-induced depletion of intracellular cGMP and Ca2+, recoverin stimulates resynthesis of cGMP. Bovine retinal recoverin has now been analyzed by electrospray mass spectrometry (ESI-MS) for post-translational modifications that might influence its activity. Heterogeneous acylation was detected at the NH2 terminus of bovine retinal recoverin. The NH2-terminal glycine of each retinal recoverin molecule is linked to one of four different types of acyl groups. The most abundant is myristoleate (14:1), but 14:0, 14:2, and 12:0 acyl residues are also present.