Molecular properties of rhodopsin and rod function

Abstract

Signal transduction in rod cells begins with photon absorption by rhodopsin and leads to the generation of an electrical response. The response profile is determined by the molecular properties of the phototransduction components. To examine how the molecular properties of rhodopsin correlate with the rod-response profile, we have generated a knock-in mouse with rhodopsin replaced by its E122Q mutant, which exhibits properties different from those of wild-type (WT) rhodopsin. Knock-in mouse rods with E122Q rhodopsin exhibited a photosensitivity about 70% of WT. Correspondingly, their single-photon response had an amplitude about 80% of WT, and a rate of decline from peak about 1.3 times of WT. The overall 30% lower photosensitivity of mutant rods can be explained by a lower pigment photosensitivity (0.9) and the smaller single-photon response (0.8). The slower decline of the response, however, did not correlate with the 10-fold shorter lifetime of the meta-II state of E122Q rhodopsin. This shorter lifetime became evident in the recovery phase of rod cells only when arrestin was absent. Simulation analysis of the photoresponse profile indicated that the slower decline and the smaller amplitude of the single-photon response can both be explained by the shift in the meta-I/meta-II equilibrium of E122Q rhodopsin toward meta-I. The difference in meta-III lifetime between WT and E122Q mutant became obvious in the recovery phase of the dark current after moderate photobleaching of rod cells. Thus, the present study clearly reveals how the molecular properties of rhodopsin affect the amplitude, shape, and kinetics of the rod response.

FIGURE 1. Targeted mutation of the rhodopsin gene in mice

A maps of W Trhodopsin gene locus, targeting vector, and targeted locus. The coding exons of the rhodopsin gene are shown as black boxes. The map of the targeting vector shows the E122Q mutation in exon 2 (*) and insertion of the neomycin-resistance gene (PGKneopA) in the middle of intron 1 in the reverse direction. The expected sizes of AvrII-generated fragments from WT (7 kb) and targeted rhodopsin (9 kb) genes detected with 5′- and 3′-flanking probes are shown. B, Southern blots of DNA from littermates. The appearance of a 9-kb fragment from the mutant allele is indicated. C, expression yields of pigments in the retinas of WT, homozygous mice with and without the positive selection maker (PGKneopA) in the genome. Retinal extracts from 10-week-old mice were prepared in the dark and the amount of pigments were estimated (see “Experimental Procedures”). D, histology of WT and mutant retinas. Eyes of 10-week-old WT (a), heterozygous (b), and homozygous littermates (c) were used. The mutant mice have the positive selection maker (PGKneopA) in the genome. 10-µm frozen sections were stained with toluidine blue. Micrographs show regions located approximately one-third of the retinal distance from the optic nerve to the ora serrata. OS, outer segment layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 10 µm.

FIGURE 2. Spectroscopic characteristics of WT rhodopsin and E122Q mutant

A, absorption spectra of WT and E122Q rhodopsins. WT and E122Q opsins (25 µm) were prepared in the respective ROS membranes suspended with buffer B (pH 7.4) and reacted with the same amounts of 11-cis-retinal (final concentration of 2.2 µm). After the reactions were completed, the pigments were bleached by a 10-min irradiation of yellow (>480 nm) light by using a Y-50 cut-off filter in the presence of 100 mm hydroxylamine. The difference spectra before and after the irradiation were then calculated. The negative peak at 360–370 nm represents the λ_max of free all-trans-retinal oxime produced by bleaching. All experiments were performed at 2 °C. B, photosensitivity of wild-type (open squares) and E122Q (closed triangles) rhodopsins extracted from respective ROS membranes with buffer A (pH 6.5). The pigments were then irradiated with a green light (500 nm) at 37 °C for adequate time in the presence of 50 mm hydroxylamine. The amounts of remaining pigments were plotted as a function of total photons exposed. C and D, estimation of the relative amount of meta-I and meta-II in the equilibrium mixtures produced by irradiation of WT (C) and E122Q (D) rhodopsins at 37 °C. WT and E122Q rhodopsins in ROS membranes (curves 1 in C and D) were suspended to buffer B (pH 7.4) and irradiated with a 500-nm light pulse and the spectra 100 ms after the irradiation of the pigments were recorded by the CCD spectrophotometer. Then the spectra (curves2 (dotted curve) in C and D) containing only meta-I and meta-II were calculated by subtracting the spectra of residual 11-cis-pigments and photo-regenerated 9-cis-pigments from the spectra recorded. The amounts of 11-cis- and 9-cis-pigments were estimated by simulation of the measured spectra at the wavelength regions longer than 580 nm (or 560 nm in E122Q sample) with those of 11-cis- and 9-cis-pigments (26, 51). The amounts of 11-cis- and 9-cis-pigments present in the illuminated WT sample were 41.8 and 4.0%, respectively. Those in the illuminated E122Q sample were 41.6 and 4.5%, respectively. The amounts of meta-I and meta-II in the mixtures were estimated by simulating the spectra with template spectra of meta-I and meta-II (26). Solid spectra in C and D are the simulated spectra of meta-I and meta-II present in the mixtures. Individual spectra of meta-I or meta-II in the mixture are also shown in C or D as broken curves. E and F, spectral changes observed in the conversion process of meta-II to meta-III in WT (E) and E122Q (F) rhodopsins. WT and E122Q rhodopsins in ROS membranes were irradiated with a 500-nm light pulse and subsequent spectral changes were monitored by the CCD spectrophotometer. Spectra were recorded 10 and 100 ms, 1, 2, 4, 8, 15, and 30 s, 1, 2, 4, and 8 min after irradiation of the sample in E, and 10 and 100 ms, 1, 2, 4, 8, 15, and 30 s, and 1 min after irradiation of the sample in F. G and H, spectral changes observed in the conversion process of meta-III to retinal plus opsin in WT (G) and E122Q (H) rhodopsins. WT and E122Q rhodopsins in ROS membranes were irradiated with a 500-nm light pulse and subsequent spectral changes were monitored by the CCD spectrophotometer. Spectra were recorded 4, 8, 15, and 30 min and 1 h after irradiation of the sample in G, and 1, 2,4, 8, 15, 30 min and 1 h after irradiation of the sample in H.

FIGURE 3. Electrophysiological recordings with a suction pipette from single rods of WT and E122Q mice

A and B, responses from WT (A) and homozygous E122Q (B) rods. In both cases, 20-ms flashes of a 500-nm light delivering 5.9, 11, 26, 46, 100, 190, 400, and 770 photons µm⁻² were given at the time instant of 0 s. Each trace is the averaged response from multiple flash trials. The records were low-pass filtered at 30 Hz. The maximal response was 12.3 pA for the WT rod and 14.9 pA for the E122Q rod. The E122Q rods were from E122Q mice without the positive selection marker (PKG neo) in the genome. C, collected action spectra of WT and E122Q rods. Sensitivity was calculated by dividing the dim-flash response amplitude by the flash intensity. Measurements were made at 450, 460, 480, 500, 510, 530, 550, 600, 650, and 680 nm. Averaged data (±S.E.) for WT (open squares; n = 9) and E122Q (closed squares; n = 6) rods. The data for E122Q rods were obtained from the rods of mice with and without the positive selection marker (PGKneopA) in the genome. For data from mice still retaining the marker, the average spectral sensitivity at 500 nm has been scaled to the same value as that for mice without the marker. D, single-photon responses of WT and E122Q rods. Thin trace is the averaged single-photon response of WT rods and the thick trace is that of E122Q rods. Averaged amplitude of the single-photon response was 0.56 pA (n = 16) for WT rods and 0.45 pA (n = 26) for E122Q rods. The amplitude of the single-photon response can be estimated from the ensemble variance-to-mean ratio of the response amplitude (see text). The E122Q rods were from the mice without the positive selection marker in the genome.

FIGURE 4. Rod response profiles simulated by the reaction scheme

Rod response profile was simulated according to Ref. with some modifications (see text).All traces are the simulated responses in which identical amounts of the rhodopsin molecules are activated at time 0. Thin and thick curves are calculated results assuming that the equilibrium constant of meta-I and meta-II are 18/82 and 34/66, respectively, and meta-II is the only intermediate that can be phosphorylated by kinase. Inset thin and thick curves are the same in the main panel. Thin and thick curves in A are calculated by assuming that meta-I is the only intermediate that can be phosphorylated by kinase. Thin and thick curves in B are calculated by assuming that both meta-I and meta-II can be phosphorylated.

FIGURE 5. Comparison of kinetics of intermediate decays with the response profiles

In these experiments the E122Q mice still retained the positive selection marker. A and B, comparison of kinetics of meta-II decay with the decline of dim-flash response in arrestin knock-out (Arr^−/−) background. A, meta-II decay time course for WT and E122Q rhodopsins in ROS membranes. ROS membranes isolated from dark-adapted mouse retinas and suspended in buffer B at 37 °C were bleached with yellow light (>520 nm-light for WT and >500 nm-light for E122Q) for 3 s. The absorbance change at 380 nm (λ_max of meta-II) was measured with a MPS-2000 spectrophotometer and plotted as a function of time after the bleach at 37 °C. Lines are single-exponential decays with a time constant of 83.3 s for WT and 9.0 s for E122Q rhodopsin. B, dim-flash responses of rods from Arr^−/− and E122Q, Arr^−/− rods. Transient peak amplitude of the response has been normalized to unity. Experimental procedures were the same as described in the legend to Fig. 3. Gray lines are single-exponential fits to current declines, with the indicated time constants. The inset compares the kinetics of the rise and the initial recovery of the responses for the WT and E122Q rods. C–E, comparison of kinetics of meta-III decay with the recovery of dark current after a bleaching light in isolated cells. C, time courses of formation and decay of meta-III for WT and E122Q rhodopsins in ROS membranes. ROS membranes were prepared from dark-adapted mouse retinas and suspended in buffer B at 37 °C. The samples were irradiated with a 500-nm light pulse and subsequent spectral changes were monitored by the CCD spectrophotometer. The changes in the difference absorbance at 460nm were then plotted as a function of time after irradiation. Curves are convolutions of two exponentials with time constants of 93 s and 14.3 min for WT rhodopsin and 3.7 s and 6.3 min for E122Q rhodopsin. The short time constant reflects the formation of meta-III from meta-II and the long time constant reflects the decay of meta-III. D, time course of recovery of dark current of a WT rod after bleaching 20% of rhodopsin. Timing of bleach is given by the arrow. Curve is a single-exponential decline with a time constant of 19.5 min. E, same experiment for a E122Q rod. Curve is a single-exponential decline with a time constant of 6.8 min.