Single amino acid residue as a functional determinant of rod and cone visual pigments

Abstract

The visual transduction processes in rod and cone photoreceptor cells begin with photon absorption by the different types of visual pigments. Cone visual pigments exhibit faster regeneration from 11-cis-retinal and opsin and faster decay of physiologically active intermediate (meta II) than does the rod visual pigment, rhodopsin, as expected, due to the functional difference between rod and cone photoreceptor cells. To identify the amino acid residue(s) responsible for the difference in molecular properties between rod and cone visual pigments, we selected three amino acid positions (64, 122, and 150), where cone visual pigments have amino acid residues electrically different from those of rhodopsin, and prepared mutants of rhodopsin and chicken green-sensitive cone visual pigment. The results showed that the replacement of Glu-122 of rhodopsin by the residue containing green- or red-sensitive cone pigment converted rhodopsin's rates of regeneration and meta II decay into those of the respective cone pigments, whereas the introduction of Glu-122 into green-sensitive cone visual pigment changed the rates of these processes into rates similar to those of rhodopsin. Furthermore, exchange of the residue at position 122 between rhodopsin and chicken green-sensitive cone pigment interchanges their efficiencies in activating retinal G protein transducin. Thus, the amino acid residue at position 122 is a functional determinant of rod and cone visual pigments.

Figure 1

Amino acid positions in which the electrical properties between the residues chicken rhodopsin and cone visual pigments differ. The transmembrane topography is based on the model of Hargrave et al. (16). Amino acid positions indicated with white circles are those in which rhodopsin and the four types of cone visual pigments have residues similar in electric properties. The gray or black circles indicate the positions at which some (gray) or almost all (black) of the cone pigments have residues electrically different from those of rhodopsin. The residues of rhodopsin replaced in this study are denoted by single-letter codes and numbered using the bovine rhodopsin numbering system. Corresponding residues of chicken green and red are also denoted.

Figure 2

Regeneration rates of wild-type and mutant rhodopsins. (A) Regeneration of wild-type, E122Q, and E122I rhodopsins monitored by change of absorbance at 530 nm. 11-cis-Retinal solution (2.5 nmol) in ethanol (5 μl) was added to the respective opsin solution (220 μl) at 2°C, and increase of absorbance at 530 nm due to the regeneration of pigment was recorded. The maximal absorbance due to the full regeneration is normalized. Solid curves are the fitted single exponential curves with time constants of 26 (wild type), 1.3 (E122Q), and 0.94 (E122I) min, respectively. (B) Rate constants of pigment regeneration in wild-type and mutant rhodopsins and native chicken rhodopsin (Rh), chicken green (cG), and chicken red (cR) (Inset). Rate constants of wild-type and native rhodopsins were normalized to 1, and the rate constants of mutant rhodopsins and native cone pigments are represented relative to those of their respective rhodopsins. The standard deviations were estimated from three independent experiments using different preparations.

Figure 3

Thermal reactions of meta II of wild-type and mutant rhodopsin. (A and B) Formation and decay of meta II monitored by change in the absorption spectrum. Wild-type (A) and E122Q (B) rhodopsins were irradiated with orange light for 30 s at 2°C, followed by continuous recording of the absorption spectra. The thin and bold lines in each panel represent the difference spectra obtained by subtracting the spectra before irradiation from those immediately after and 20 min after irradiation, respectively. The spectra shown in the Insets are the difference spectra between thin and bold lines. (Bars = 0.003 absorbance unit.) (C) Course of conversion from meta II to meta III in wild-type, E122Q, and E122I rhodopsins. Increase in absorbance at 460 nm due to the formation of meta III from meta II is plotted as a function of incubation time after the irradiation. Solid curves are the fitted single exponential curves with the time constants of 190 (wild type), 3.3 (E122Q), and 12 (E122I) min, respectively. (D) Rate constants of meta II decay in wild-type and mutant rhodopsins, and native chicken rhodopsin (Rh), chicken green (cG), and chicken red (cR) (Inset). Rate constants of wild-type and native rhodopsins were normalized to 1, and the rate constants of mutant rhodopsins and native cone pigments are represented relative to those of their respective rhodopsins. The standard deviations were estimated from three independent experiments using different preparations.

Figure 4

Change in the activation of transducin by native, wild-type, and mutant rhodopsin and chicken green. The pigments were added to the reaction mixture containing transducin 6 min after irradiation, and the extents of GTPase activity were measured. The relative activities to those immediately after irradiation were plotted. The standard deviations were estimated from four independent experiments using different preparations.