Retinal rods respond to light with a membrane hyperpolarization produced by a G-protein-mediated signalling cascade that leads to cyclic GMP hydrolysis and the consequent closure of a cGMP-gated channel that is open in darkness. A protein that forms this channel has recently been purified from bovine retina and molecularly cloned, suggesting that the native cGMP-gated channel might be a homo-oligomer. Here we report the cloning of another protein from human retina which has only about 30% overall identity to the rod channel subunit. This protein, immunocytochemically localized to rod outer segments, does not form functional channels by itself. However, when co-expressed with the cloned human rod channel protein, it introduces rapid flickers to the channel openings that are characteristic of the native channel. The hetero-oligomeric channel is also highly sensitive to the blocker L-cis-diltiazem, like the native channel. This new protein thus seems to be another subunit of the native rod channel. The hetero-oligomeric nature of the rod channel means that it is no exception to a common motif shared by other ligand-gated channels.

Retinitis pigmentosa (RP) is a prevalent cause of blindness caused by a large number of different mutations in many different genes. The mutations result in rod photoreceptor cell death, but it is unknown why cones die. In this study, we tested the hypothesis that cones die from oxidative damage by performing immunohistochemical staining for biomarkers of oxidative damage in a transgenic pig model of RP. The presence of acrolein- and 4-hydroxynonenal-adducts on proteins is a specific indicator that lipid peroxidation has occurred, and there was strong immunofluorescent staining for both in cone inner segments (IS) of two 10-month-old transgenic pigs in which almost all rods had died, compared to faint staining in two 10-month-old control pig retinas. In 22- and 24-month-old transgenic pigs in which all rods and many cones had died, staining was strong in cone axons and some cell bodies as well as IS indicating progression in oxidative damage between 10 and 22 months. Biomarkers for oxidative damage to proteins and DNA also showed progressive oxidative damage to those macromolecules in cones during the course of RP. These data support the hypothesis that the death of rods results in decreased oxygen consumption and hyperoxia in the outer retina resulting in gradual cone cell death from oxidative damage. This hypothesis has important therapeutic implications and deserves rapid evaluation.

Mutations in the genes encoding two proteins of the retinal rod phototransduction cascade, opsin and the j3 subunit of rod cGMP phosphodiesterase, cause retinitis pigmentosa (RP) in some families. Here we report defects in a third member of this biochemical pathway in still other patients with this disease. We screened 94 unrelated patients with autosomal dominant RP and 173 unrelated patients with autosomal recessive RP for mutations in the gene encoding the a subunit of the rod cGMP-gated cation channel. Five mutant sequences cosegregated with disease among four unrelated families with autosomal recessive RP. Two of these were nonsense mutations early in the reading frame (Glu76End and Lysl39End) and one was a deletion encompassing most if not all of the transcriptional unit; these three alleles would not be expected to encode a functional channel. The remaining two mutations were a missense mutation (Ser316Phe) and a frameshift [Arg654(1-bp del)] mutation truncating the last 32 aa in the C terminus. The latter two mutations were expressed in vitro and found to encode proteins that were predominantly retained inside the cell instead ofbeing targeted to the plasma membrane. We conclude that the absence or paucity of functional cGMP-gated cation channels in the plasma membrane is deleterious to rod photoreceptors and is an uncommon cause of RP.Retinitis pigmentosa (RP) is a genetically heterogeneous set of diseases in which affected individuals develop progressive degeneration of the rod and cone photoreceptors. Patients typically experience night blindness by age 20 followed by progressive loss of peripheral visual field and later central visual field that leads to blindness usually in middle age. Oral vitamin A supplementation slows the course of the disease in most cases (1). Dominant, recessive, X chromosome-linked, and digenic patterns of inheritance are exemplified by families with RP, and even among the families with the same inheritance pattern, there is nonallelic heterogeneity. More than 15 genes have been implicated by linkage studies, five of which have been identified to date. Two of these genes encode proteins known to function in the phototransduction pathway [namely, rhodopsin (2) and the ,3 subunit of rod cGMPphosphodiesterase (3)], two are photoreceptor-specific proteins of unknown function [peripherin/RDS (4-6) and ROM1 (6)], and one is an unconventional myosin (7).Here we report the analysis of the gene encoding the a subunit of the rod cGMP-gated cation channel, which is the protein involved in the last stage of the phototransduction pathway (for review, see ref. 8). The rod cGMP-gated cation channel is a heterooligomer composed of two homologous subunits (a and (3), each with cytoplasmic N and C termini, six putative transmembrane domains, and a pore region (9-11).

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