Fig. 4. Rod cyclic nucleotide-gated (CNG) channels interact with peripherin, a Na+/K+,Ca2+ exchanger, and a protein tyrosine kinase (PTK). See text for details. ABCR ATP-binding cassette.

photoreceptors are components of even larger protein complexes. In both rods and cones, CNG channels are intimately associated with the Na+/K+,Ca2+ exchanger via their A subunits [44, 48]. The juxtaposition of the channel and the exchanger suggests that Ca2+ dynamics inside the cell are localized to microdomains in the vicinity of the channel (Fig. 4).

An unexpected finding suggests that CNG channels in photoreceptors are also associated with a protein tyrosine kinase (PTK). Genistein, an inhibitor of PTKs, reversibly slows the gating kinetics and reduces the maximal current responses of native CNG channels in the absence of ATP (i.e., independent of phosphorylation) [49]. The effect of genistein on CNG channels is ameliorated by other PTK inhibitors that do not otherwise affect CNG channels. In conclusion of this finding, it was suggested that genistein binds to the PTK rather than the CNG channel. It is thought that PTK undergoes conformational changes on binding of genistein that are transferred to the tightly associated CNG channel, thereby modifying channel function. At this time, however, a physical association between PTKs and CNG channels has not been demonstrated.

MODULATION BY PHOSPHORYLATION AND ALL-TRANS RETINAL

The cGMP sensitivity of photoreceptor CNG channels is modulated by tyrosine and serine/threonine phosphorylation. The effects of tyrosine phosphorylation on rod CNG channels have been extensively studied in vitro [24, 49]. Heteromeric CNGA1/CNGB1 channels expressed in Xenopus oocytes show a roughly twofold increase in cGMP sensitivity developing over time after patch excision. This increase was attributed to the spontaneous dephosphorylation of single tyrosine residues in the cNMP-binding site of both CNGA1 and CNGB1 by protein tyrosine phosphatases (PTPs). A signaling pathway that may regulate tyrosine phosphorylation of rod CNG channels in vivo involves insulin-like growth factor 1 (IGF-1) [49]. IGF-1 is released from the pigment epithelium and increases the cGMP sensitivity of CNG channels in rod photoreceptors. IGF-1 is thought to exert the effect on CNG channels through the regulation of a PTP.

Besides tyrosine phosphorylation, also serine/threonine phosphorylation has been implicated in the regulation of the ligand sensitivity of retinal CNG channels. Repeated dose-response measurements with CNG channels in excised patches from rod outer segments revealed a slow increase in cGMP sensitivity over time, characterized by an up to tenfold decrease in the EC50 (cGMP) [50]. The enhancement of ligand sensitivity was slowed by ATP or inhibitors of serine/threonine phosphatases and accelerated by purified type I phosphatase. Similarly, a threefold decrease in cGMP sensitivity was observed for heterologously expressed CNGA3 channels on exposure to phorbol esters, substances that stimulate protein kinase C (PKC) [51]. This effect is due to the phosphorylation of two serine residues, also located in the cNMP-binding site.

The cGMP sensitivity of cone CNG channels, at least in chicken, is under the control of a circadian rhythm [52]. During the subjective night, the sensitivity is approximately twofold higher than during the subjective day. This circadian modulation is apparently driven by rhythms in the activities of the extracellular receptor kinase (ERK) form of mitogen-activated protein kinase and the Ca2+/calmodulin-dependent protein kinase II (CaMKII), but the detailed mechanism underlying this control is unknown.

Besides phosphorylation, rod CNG channels are also inhibited by the phospholipid Phosphoinositoldiphosphate(PIP2)andall-transretinal,thephotoisomerizedpigmentreleased from rhodopsin after light activation [53]. All-trans retinal directly inhibits the homomeric CNGA1 and heteromeric CNGA1/CNGB1 channels at nanomolar concentrations, probably due to a decrease in the open probability. The effects observed in a heterologous expression system might also apply to native rod CNG channels, when bright illumination is expected to lead to elevated concentrations of photoisomerized pigment in the outer segment.

SYNTHESIS, MATURATION, AND TARGETING OF CNG CHANNELS

The predominant form of CNGA1 in bovine rod outer segments has an apparent molecular mass of 63kDa, which is significantly less then the molecular mass predicted from the full-length complementary DNA or the apparent molecular mass of heterologously expressed CNGA1 (78 kDa). This discrepancy in size is due to a photoreceptor-specific proteolytic process by which the 92 N-terminal amino acids of CNGA1 are removed [28]. Similar processing of the rod A subunits seems to be common across species, and at least the chicken cone A subunit also undergoes posttranslational cleavage [14, 54]. Another posttranslational modification of CNGA1 and CNGA3 is the glycosylation of an asparagine residue in the extracellular loop between S5 and the p region [29, 55]. In contrast to the CNG channel A subunits, CNGB1 and CNGB3 are unglycosylated. Currently, the functions of proteolytic cleavage and glycosylation are unknown since neither affects channel activity. The posttranslational modification might be required for channel targeting or for the interaction of CNG channels with other photoreceptor proteins.

For proper targeting, CNG channels need to be translocated from their site of synthesis in the membrane of the endoplasmic reticulum (ER) within the main cell body to the plasma membrane of the outer segment. Impaired plasma membrane targeting in heterologous expression systems has been reported for a variety of mutant CNGA1 and CNGA3 subunits found in patients with either rod or cone dysfunction, respectively. Most of these mutants fold improperly and thus fail to leave the ER [55–57].

In a naturally occurring CNGA1 mutant with an amino acid substitution and truncation C-terminal to the cNMP-binding site (R654D-stop in human CNGA1), a more specific mechanism of ER retention seems to apply. Two studies investigated the mechanism of ER retention for this mutant, but with conflicting results. One study found that the mutation generates an ER retention signal itself, while another study provided evidence that the mutated CNGA1 is unable to mask an ER retention signal in CNGB1 [58, 59]. At present it is unclear how the conflicting results can be reconciled.

A glimpse of how CNG channels are transported into the outer segment was obtained in a study using subunits of the olfactory CNG channels (CNGA2 and CNGB1b, a splice variant of CNGB1 that lacks the GARP domain) expressed in a ciliated cell line [60]. In this study, the ciliary transport of CNGA2 is dependent on CNGB1b and, more specifically, on a short sequence of four amino acids within this subunit (RVxP, with x representing a variable amino acid). A similar mechanism might apply to rod CNG channels and could explain why rods from mice lacking CNGB1 have decreased levels of CNGA1 in the outer segment [47].

VISUAL DYSFUNCTION CAUSED BY MUTANT CNG CHANNEL GENES

Mutations in the genes for CNG channels of photoreceptors are associated with the hereditary visual diseases retinitis pigmentosa (RP), achromatopsia, and cone dystrophy [16, 17, 57, 61–68] (see also Appendix). Retinitis pigmentosa is a clinically and genetically heterogeneous group of diseases characterized by night blindness, a progressive loss of the peripheral visual field, and eventual loss of central vision, resulting in blindness. These symptoms reflect early dysfunction and degeneration of rod photoreceptors, followed by a slower degeneration of cone photoreceptors that proceeds from the periphery to the center of the visual field. Mutations in the CNGA1 or CNGB1 gene only account for a few percent of autosomal recessive RP. Achromatopsia is a recessive, nonprogressive disease resulting from the dysfunction of cone photoreceptors. Symptoms include absence of color vision, light sensitivity (photophobia), and poor visual acuity. Of all cases of achromatopsia, 20–30% are caused by CNGA3 mutations and 40–50% by mutations in CNGB3 [69]. Moreover, in some instances, mutations in CNGA3 or CNGB3 result in cone dystrophy, a disease related to achromatopsia but characterized by the progressive loss of cone function and sometimes the progressive loss of rod function. The vast number of deleterious single amino acid substitutions in CNGA3 (Fig. 5) indicates that there is little tolerance for sequence variations in CNG channel A subunits with respect to photoreceptor function. Mutations do not, however, necessarily lead to nonfunctional CNG channels. Some mutant channel proteins, in particular those exclusively found in patients with residual photoreceptor function (Fig. 5, underlined mutations), are expected to form channels, although with functional alterations.

A mild form of achromatopsia with considerably preserved cone function is caused

by the heterozygous mutations T224R and T369S in CNGA3 (A3T224R and A3T369S) [26]. The patients are able to discriminate saturated but not desaturated colors. Psychophysical

and electroretinographical analyses showed that the cone system is characterized by lower light sensitivity and perturbed signal transfer from cones to postsynaptic neurons. Patch-clamp analysis of heterologously expressed subunits revealed that only A3T369S

Fig. 5. Mutations in cone photoreceptor B subunit (CNGA3) are associated with cone dysfunction. See text for details. Underlined amino acid substitutions are found in patients with residual cone function. cNMP nucleoside 3′,5′-cyclic monophosphate, Fs frame shift mutation, del deletion.

produces functional channels, although with grossly altered permeation of monovalent and divalent ions, gating, and ligand sensitivity. Coexpression of wild-type CNGB3 with A3T369S restored most of the native properties, except for the altered Ca2+ permeation. The properties of A3T369S/CNGB3 channels suggest that the mild form of achromatopsia results from relatively subtle changes in ion flux through the cone CNG channel.

Another channel defect appears to be common in patients with cone dysfunction.

Several missense mutations associated with cone dystrophy (A3N471S, A3R563H, B3R403Q) or complete achromatopsia (B3F525N, B3D633G, and the Pingelap mutation B3S435F) have been reported to produce channels with moderately increased cGMP sensitivity [25, 66, 70,

71]. These findings are surprising given the severe cone defects in the patients but might reflect the fact that the functional integrity of photoreceptors relies on a precisely tuned transduction machinery. How a similar functional defect in different mutant channels can produce different clinical phenotypes remains unresolved.

Intriguingly, the absence or malfunction of CNG channels often leads to photoreceptor loss and even retinal degeneration. Our understanding of the trigger, the detailed mechanisms underlying retinal degeneration, and in particular the role of CNG channels in these processes is incomplete. We have to assume that the survival of photoreceptors critically depends on the structural and functional integrity of their enzymatic machinery. The discovery of efficient CNG channel blockers (e.g., [72]) opens an avenue for the treatment of retinal diseases caused by abnormally high CNG channel activity.