Signaling states of rhodopsin. Formation of the storage form, metarhodopsin III, from active metarhodopsin II

Abstract

Vertebrate rhodopsin consists of the apoprotein opsin and the chromophore 11-cis-retinal covalently linked via a protonated Schiff base. Upon photoisomerization of the chromophore to all-trans-retinal, the retinylidene linkage hydrolyzes, and all-trans-retinal dissociates from opsin. The pigment is eventually restored by recombining with enzymatically produced 11-cis-retinal. All-trans-retinal release occurs in parallel with decay of the active form, metarhodopsin (Meta) II, in which the original Schiff base is intact but deprotonated. The intermediates formed during Meta II decay include Meta III, with the original Schiff base reprotonated, and Meta III-like pseudo-photoproducts. Using an intrinsic fluorescence assay, Fourier transform infrared spectroscopy, and UV-visible spectroscopy, we investigated Meta II decay in native rod disk membranes. Up to 40% of Meta III is formed without changes in the intrinsic Trp fluorescence and thus without all-trans-retinal release. NADPH, a cofactor for the reduction of all-trans-retinal to all-trans-retinol, does not accelerate Meta II decay nor does it change the amount of Meta III formed. However, Meta III can be photoconverted back to the Meta II signaling state. The data are described by two quasi-irreversible pathways, leading in parallel into Meta III or into release of all-trans-retinal. Therefore, Meta III could be a form of rhodopsin that is stored away, thus regulating photoreceptor regeneration.

Reaction 1

Reaction 2

Fig. 1. Change of intrinsic fluorescence monitoring the decay of photoactivated rhodopsin (R*) in bovine rod disk membranes

A, kinetics of fluorescence increase (a) after saturating photo-activation of rhodopsin (1 μm) in disk membranes (λ_ex = 295 nm, λ_em = 330 nm, pH 6.5, 20 °C). The amount of protonated Schiff base left at the indicated times (b) was estimated from the absorption at 440 nm after acid denaturation (pH 1.9) and solubilization of the samples. B, fluorescence emission spectra (λ_ex = 295 nm) of rhodopsin membranes (pH 6.5, 20 °C) in the dark (a), immediately after photoactivation (b) and after 60 min (c).

Fig. 2. Temperature dependence of fluorescence change

A, fluorescence increase after photoactivation of rhodopsin (1 μm) at different temperatures (pH 6.5). B, Arrhenius plot of the rates of fluorescence increase. Solid line is a linear least squares fit to the data.

Fig. 3. pH dependence of fluorescence change

Families of fluorescence traces measured at 20 °C and the indicated pH in the absence (A) and in the presence (B) of 100 μm G_tα-HAA. Samples (1 μm rhodopsin in disk membranes) were illuminated at pH 6.0 for 15 s and immediately adjusted to the respective pHs. Insets, fluorescence traces measured at 33 °C and pH 6.4 (red traces) and pH 8.1 (black traces). C, pH dependence of the maximum amplitude of the fluorescence increase (20 °C) with and without G_tα-HAA (open and closed circles, respectively). Solid lines are fits to the titration data using linear hyperbolic functions. The difference between the fitted curves yield the pH dependence of Meta III formation (inset). D, pH dependence of the reaction rates of fluorescence increase (20 °C) with and without G_tα-HAA (open and closed circles, respectively).

Fig. 4. Effect of enzymatic reduction of all-trans-retinal on the changes in fluorescence

Fluorescence increase of illuminated disk membranes (pH 7.5, 20 °C) in the absence of NADPH (black), in the presence of 70 μm NADPH (red), and in the presence of both 70 μm NADPH and 100 μm G_tα-HAA (blue), respectively.

Fig. 5. UV-visible difference spectra recorded during decay of Meta II with concurrent regeneration of opsin to rhodopsin with 11-cis-retinal

Time course of spectral changes during rhodopsin decay and regeneration at pH 7.5 and 33 °C without NADPH (A), with 70 μm NADPH (B), and with both G_tα -HAA-peptide (100 μm) and NADPH (70 μm) (C). Disk membranes (4.3 μm rhodopsin) were fully bleached at pH 6.0 and adjusted immediately to pH 7.5. After addition of equimolar 11-cis-retinal, the absorption was set to zero. The difference spectra were taken at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 10, 12, 14, and 15 min, respectively.

Fig. 6. Characterization of Meta III by FTIR spectroscopy

FTIR difference spectra were recorded immediately after Meta II formation (Meta II minus rhodopsin) and after the times indicated (decay products minus rhodopsin) at 23 °C, pH 7.5 (A) and pH 5.9 (B). A second illumination with green light after 120 min yields the difference spectra between a form of Meta II (see text and Ref. 37) and Meta III (Meta II-like minus Meta III).

Fig. 7. Characterization of Meta II decay products by UV-visible spectroscopy

The measurements were carried out as described under “Experimental Procedures.” A, spectrophotometric characterization of the formation and decay of Meta I/II. Absorption spectrum of a disk membrane suspension in the dark (a) and after 15 s of illumination at pH 6.0 (b). After adjusting the sample to pH 8.0 by addition of NaOH, further spectra were recorded 1.5 (c), 5 (d), 15 (e), and 35 (f) min after illumination of the suspension. Note the shift of the baseline upon addition of NaOH. B, after complete decay of Meta II (40 min) 100 μm G_tα-HAA was added to the sample shown in A, and spectra were taken before (a) and after (b) a second illumination of the suspension with green light. A second sample was allowed to decay at pH 8.0 in the presence of 100 μm G_tα-HAA-peptide, and a spectrum was taken after 45 min (c).

Fig. 8. Model of rhodopsin

The model is based on a high resolution crystal structure (1HZX) (56). a, rhodopsin with indicated positions of Trp residues. b, close-up of the vicinity of Trp¹²⁶. c, close-up of the vicinity of Trp²⁶⁵.