Light-induced exposure of the cytoplasmic end of transmembrane helix seven in rhodopsin

A key step in signal transduction in the visual cell is the light-induced conformational change of rhodopsin that triggers the binding and activation of the guanine nucleotide-binding protein. Site-directed mAbs against bovine rhodopsin were produced and used to detect and characterize these conformational changes upon light activation. Among several antibodies that bound exclusively to the light-activated state, an antibody (IgG subclass) with the highest affinity (Ka ≈ 6 × 10−9 M) was further purified and characterized. The epitope of this antibody was mapped to the amino acid sequence 304–311. This epitope extends from the central region to the cytoplasmic end of the seventh transmembrane helix and incorporates a part of a highly conserved NPXXY motif, a critical region for signaling and agonist-induced internalization of several biogenic amine and peptide receptors. In the dark state, no binding of the antibody to rhodopsin was detected. Accessibility of the epitope to the antibody correlated with formation of the metarhodopsin II photointermediate and was reduced significantly at the metarhodopsin III intermediate. Further, incubation of the antigen–antibody complex with 11-cis-retinal failed to regenerate the native rhodopsin chromophore. These results suggest significant and reversible conformational changes in close proximity to the cytoplasmic end of the seventh transmembrane helix of rhodopsin that might be important for folding and signaling.

Inhibition of the visual cycle in vivo by 13-cis retinoic acid protects from light damage and provides a mechanism for night blindness in isotretinoin therapy

Isotretinoin (13-cis retinoic acid) is frequently prescribed for severe acne [Peck, G. L., Olsen, T. G., Yoder, F. W., Strauss, J. S., Downing, D. T., Pandya, M., Butkus, D. & Arnaud-Battandier, J. (1979) N. Engl. J. Med. 300, 329–333] but can impair night vision [Fraunfelder, F. T., LaBraico, J. M. & Meyer, S. M. (1985) Am. J. Ophthalmol. 100, 534–537] shortly after the beginning of therapy [Shulman, S. R. (1989) Am. J. Public Health 79, 1565–1568]. As rod photoreceptors are responsible for night vision, we administered isotretinoin to rats to learn whether night blindness resulted from rod cell death or from rod functional impairment. High-dose isotretinoin was given daily for 2 months and produced systemic toxicity, but this caused no histological loss of rod photoreceptors, and rod-driven electroretinogram amplitudes were normal after prolonged dark adaptation. Additional studies showed, however, that even a single dose of isotretinoin slowed the recovery of rod signaling after exposure to an intense bleaching light, and that rhodopsin regeneration was markedly slowed. When only a single dose was given, rod function recovered to normal within several days. Rods and cones both showed slow recovery from bleach after isotretinoin in rats and in mice. HPLC analysis of ocular retinoids after isotretinoin and an intense bleach showed decreased levels of rhodopsin chromophore, 11-cis retinal, and the accumulation of the biosynthetic intermediates, 11-cis and all-trans retinyl esters. Isotretinoin was also found to protect rat photoreceptors from light-induced damage, suggesting that strategies of altering retinoid cycling may have therapeutic implications for some forms of retinal and macular degeneration.