|
|
|
|
|
|
|
||
ARTICLE |
Correspondence to Yoshinori Shichida: shichida{at}vision-kyoto-u.jp
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
11% of rhodopsin in wild-type retina. Single-cell recordings of wild-type and homozygous rods suggested that the flash sensitivity and the single-photon responses from mouse green were three to fourfold lower than those from rhodopsin after correction for the differences in cell volume and levels of several signal transduction proteins. Subsequent measurements using heterozygous rods expressing both mouse green and rhodopsin E122Q mutant, where these pigments in the same rod cells can be selectively irradiated due to their distinctive absorption maxima, clearly showed that the photoresponse of mouse green was threefold lower than that of rhodopsin. Noise analysis indicated that the rate of thermal activations of mouse green was 1.7 x 10–7 s–1, about 860-fold higher than that of rhodopsin. The increase in thermal activation of mouse green relative to that of rhodopsin results in only 4% reduction of rod photosensitivity for bright lights, but would instead be expected to severely affect the visual threshold under dim-light conditions. Therefore, the abilities of rhodopsin to generate a large single photon response and to retain high thermal stability in darkness are factors that have been necessary for the evolution of scotopic vision.
H. Imai's present address is Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Aichi 484-8506, Japan.
Y. Ueda's present address is Nagahama City Hospital, Nagahama, Shiga 526-8380, Japan.
Abbreviations used in this paper: ES, embryonic stem; GRK, G protein–coupled receptor kinase; GTP
S, guanosine 5'-O-(3-thiotriphosphate); ROS, rod outer segment.
 |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Ebrey and Koutalos, 2001; Arshavsky et al., 2002). Previously, amino acid sequences of rod and cone visual pigments have been determined, and phylogenetic analyses concluded that an ancestral visual pigment evolved first into four groups of cone visual pigments and then the group of rod visual pigments, rhodopsin, diverged from one of the cone visual pigment groups (Okano et al., 1992). Although there are a few exceptions, rhodopsin and cone visual pigments are expressed in rods and cones, respectively. These facts suggest that rhodopsin would have acquired unique molecular properties in the course of molecular evolution so as to meet the requirement of scotopic vision where simultaneous photon absorptions by rods occur at a very low rate (Hecht et al., 1942; Barlow, 1956).
Rods are able to respond to single-photon stimuli (Baylor et al., 1979). The amplitude of the single photon response of a photoreceptor cell is dependent on how efficiently the phototransduction cascade is activated by the visual pigment. Comparison of mouse rod and cone photoresponses by means of single cell recording has demonstrated that the amplification efficiency of the transduction cascade per activated visual pigment is at least fivefold higher in rods than in cones (Nikonov et al., 2006). Therefore, it is of interest to elucidate the contributions of rhodopsin and cone pigments to the differences in amplification efficiencies of rods and cones.
Electrophysiological studies have also demonstrated that rods exhibit a very low level of noise in the dark (Baylor et al., 1980, 1984). This property should be due in part to the extremely inert character of rhodopsin in the dark state; that is, thermal activation of a single rhodopsin molecule in amphibian rod occurs spontaneously only about once every 3,000 years at room temperature. In contrast, the dark noise of cones, especially that of red-sensitive salamander cones, is considerably higher than that of rods, and originates predominantly from spontaneous thermal activations of red-sensitive cone visual pigments (Rieke and Baylor, 2000; Sampath and Baylor, 2002). A recent study of Xenopus rods expressing red-sensitive cone pigments has demonstrated that the low thermal stability of red-sensitive cone visual pigments activated phototransduction in the dark, thereby resulting in reduced sensitivity and accelerated response kinetics through "light adaptation" (Kefalov et al., 2003). These results suggest that the thermal stability of visual pigment in the dark is a significant factor in determining the characteristics of rods and cones. However, for blue-sensitive and green-sensitive cones, the contribution of thermal stability of visual pigment is not prominent, because the variance in the dark current arose mostly from a higher frequency biological noise component (continuous noise) that could be attributed to a molecular source downstream of the pigment (Lamb and Simon, 1977; Rieke and Baylor, 2000; Holcman and Korenbrot, 2005). Moreover, cone visual pigments in amphibian retinas widely studied by electrophysiology contain mostly an A2 retinal as their chromophore and A2-based pigments are expected to be less stable than the A1-based visual pigments, which are used in mammalian retinas (Donner et al., 1990). Therefore, it is desirable to examine whether the thermal stability of cone visual pigments really affects the response properties of mammalian photoreceptor cells.
In the present study, we generated knock-in mice in which the endogenous rhodopsin was replaced with mouse green-sensitive cone visual pigment (mouse green) in order to investigate the contribution of visual pigments to photoresponse properties. Mouse green exhibits an absorption maximum at 510 nm, which belongs to the long wavelength–sensitive group (L group) of visual pigments that includes the human red and salamander red (Okano et al., 1992; Ebrey and Koutalos, 2001). We compared the dim-light responses and the dark noise of the knock-in rods with those of wild-type rods using suction pipette recording. In addition, to directly evaluate the contribution of visual pigment properties, we attempted to compare single photon responses generated by mouse green and rhodopsin in the same photoreceptor cells. For this purpose, we prepared heterozygous mice that have rod photoreceptor cells containing mouse green and E122Q rhodopsin, the latter of which exhibits an absorption maximum that was blue shifted by 23 nm from that of mouse green, but which generated a photoresponse similar to that of wild-type rhodopsin (Imai et al., 2007). The results showed that the amplitude of the single photon response elicited by mouse green was about one third of that elicited by wild-type rhodopsin. We also found that mouse green exhibited a rate of thermal activations in darkness that was 1.7 x 10–7 s–1, 860-fold larger than for mouse rhodopsin. These results are discussed in relation to the different photoresponse properties between rods and cones.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) derived from pCIneo (Promega) that was present on a 95-bp blunt fragment. The targeting vector was transferred into CMTI-1 embryonic stem (ES) cells (Cell and Molecular Technologies) by electroporation and individual neomycin-resistant clones were isolated and expanded in 48-well plates. Properly targeted ES cells were selected by Southern blot hybridized with a 5' flanking probe and injected into 3.5-d C57BL/6J blastocysts. Chimeric mice were then bred to obtain mice in which one or both alleles of rhodopsin locus were altered. For some experiments, mouse green knock-in mice (mG/mG) were crossed with E122Q knock-in (RhEQ/RhEQ) mice in which the rhodopsin gene contained the E122Q rhodopsin mutation (Imai et al., 2007). All the experimental procedures were performed in accordance with the guidelines set by Kyoto University, and were approved by the local committee for the handling of experimental animals in the Graduate School of Science, Kyoto University. Genotyping of knock-in lines was performed by Southern blot analysis. In brief, genomic DNA was prepared from mouse tails, and 1 µg of DNA was digested overnight with BamHI, electrophoretically fractionated in a 0.75% agarose gel, denatured, and transferred to a nylon membrane (Roche). Hybridization was done overnight using the standard protocol for the DIG-labeled DNAs. The chemiluminesence signal derived from CDP-Star (Roche) was detected by X-ray film.
The transcript was analyzed by RT-PCR from a retinal extract of 4-wk-old mice prepared using RNeasy Mini kit (QIAGEN). After digestion of genomic DNA by DNaseI, the first strand cDNA was synthesized with a reverse primer, RhC11 (5'-TGTCATGTTCCTGATA-3'), complementary to the sequence immediately preceding the first poly(A) addition signal of mouse rhodopsin. The cDNA products from endogenous rhodopsin and recombinant mouse green genes were amplified by using the rhodopsin 5' UTR (5'-AGCAGCCTTGGTCTCTGTCT-3') and 3' UTR (5'-GTGGATGGATGTCCTTTGTC-3') primer pairs (Kodama et al., 2005).
Estimation of Protein Expression Level
Protein expression levels were estimated by spectroscopy and Western blotting. Retinas were isolated from mouse eyes, homogenized, and extracted with buffer E (1% n-dodecyl-ß-D-maltoside, 140 mM NaCl, 50 mM HEPES, pH 6.5) for spectroscopy or with SDS-PAGE sample buffer (8% SDS, 0.125 M Tris/Cl [pH 6.8], 20% glycerol, 5% mercaptoethanol) for Western blotting. The concentration of visual pigment in the extract solubilized with buffer E was estimated from the maximal absorbance of a difference spectrum before and after irradiation with >500-nm light in the presence of hydroxylamine and using molar extinction coefficients of both rhodopsin and mouse green at their absorption maxima (502 and 510 nm, respectively) of 40,200 M–1 cm–1 (Imai et al., 2007) and 45,500 M–1 cm–1 (Onishi et al., 2005), respectively. The concentration of hydroxylamine in the rhodopsin extract from wild-type retinas was 100 mM, while that in the mouse green from mG/mG retinas was 10 mM. This is because mouse green is more sensitive to hydroxylamine than rhodopsin (Okano et al., 1989; Johnson et al., 1993) and it was stable only in the presence of <10 mM hydroxylamine.
The ratio of the two kinds of pigments in the heterozygous retinas can be estimated by monitoring the bleaching processes of these pigments in the presence of 200 mM hydroxylamine in the dark. Hydroxylamine bleaches these pigments by reacting with their retinylidene Schiff base chromophores, but the reaction rate is considerably faster in mouse green than in rhodopsin (Okano et al., 1989; Johnson et al., 1993). The retinal extracts were incubated in the presence of 200 mM hydroxylamine for 12 h at 20°C and then it was irradiated with >500-nm light for 5 min to completely bleach the remaining pigments in the extract. The bleaching processes for wild-type or homozygous retinas were monitored at the 
max of the relevant visual pigments and were fit by a single-exponential function to estimate the time constants of hydroxylamine reaction. For the Rh/mG mice, retinal extracts from five mice were incubated with hydroxlamine and the bleaching process was monitored at 500 nm. The bleaching kinetics was fit with a double-exponential function using the estimated reaction time constants of rhodopsin and mouse green determined above. The ratio of rhodopsin and mouse green was estimated from the fitting results corrected for the different molecular extinction coefficients at 500 nm (40,200 M–1 cm–1 for rhodopsin; 43,900 M–1 cm–1 for mouse green). For the RhEQ/mG mice, retinal extracts from five mice were incubated with hydroxylamine, and the bleaching process was monitored at 498 nm, which was the isosbestic point of the spectra of E122Q rhodopsin and mouse green. The bleaching kinetics was fit with a double-exponential function using the estimated reaction time constants of E122Q rhodopsin and mouse green.
The amount of other signal transduction proteins was determined by Western blotting. Retinas were removed from the eyes, placed in 250-µl SDS-PAGE sample buffer, and homogenized by sonication for 10 s with a sonicator (UR-20P; TOMY SEIKO). The homogenate was subjected to electrophoresis on 12.5% polyacrylamide gels and transferred onto polyviniliden difluoride membrane for 1–1.5 h. As a standard, four diluted samples prepared from a wild-type retina homogenate were run in neighboring lanes on the same gel. After blocking with 5% (weight/volume) ECL blocking agent (GE Healthcare) in PBS (140 mM NaCl, 2.6 mM KCl, 1.8 mM KH2PO4, 8.1 mM Na2HPO4) containing 0.05% Tween 20, the proteins in the membrane were probed with primary antibodies. The antibodies were raised against the rod transducin 
-subunit (Suzuki et al., 1993), PDE- (0.2 µg/ml; Affinity BioReagents) and PDEß- (0.2 µg/ml; Affinity BioReagents) subunits, GRK1 (1 µg/ml; Santa Cruz Biotechnology), CNG1 (1:15 dilution; PMc101; Korschen et al., 1995), rhodopsin 1D4 (1:10,000 dilution), and mouse green opsin (1:10,000 dilution, Medical and Biological Laboratories, custom antibody against N-terminal peptide of mouse green AQRLTGEQTLDHYEDS sequence; Applebury et al., 2000). Horseradish peroxidase–conjugated secondary antibodies were used at a dilution of 1:20,000, and protein bands were visualized by ECL (GE Healthcare). Integrated densities of scanned individual bands were measured with Scion Image software (Scion) and the densities of mutant samples were compared with standard intensities of the wild-type samples to estimate the relative amounts of the proteins.
Photosensitivities of Visual Pigments
Mouse rhodopsin, E122Q rhodopsin mutant (E122Q rhodopsin), and mouse green were extracted with buffer E from mouse retinas of dark-adapted wild-type, RhEQ/RhEQ, and mG/mG mice, respectively. The pigments were irradiated with 500-nm light in the presence of 100 mM hydroxylamine for rhodopsin and E122Q rhodopsin, and in the presence of 5 mM hydroxylamine for mouse green at 2°C. The light intensity was monitored with a photodiode (S1226-5BQ; Hamamatsu Photonics). The amount of pigment in the extracts was estimated from the difference spectra calculated by subtracting spectra obtained after complete bleaching by irradiation with >500-nm light from those obtained before irradiation.
Histology
Eyes were removed and fixed for 4 h in 4% paraformaldehyde in PBS at 4°C. After cryoprotection in cold 30% sucrose, the tissue was mounted with OCT compound (Tissue-Tek) and sectioned at 10-µm thickness along the vertical meridian passing through the optic nerve. Immunostaining was performed using methods described previously (Onishi et al., 2005).
For electron microscopy, whole eyeballs were hemisected and their posterior parts including the retinas were prefixed with 2% glutaraldehyde and 2% paraformaldehyde in the buffer consisting of 30 mM HEPES, 100 mM NaCl, and 2 mM CaCl2 (pH 7.4) for 2 h at room temperature. They were then post-fixed with 1% OSO4 in the same buffer for 1 h in a conventional way (Usukura and Yamada, 1987). Fixed samples were dehydrated gradually in ascending concentrations of ethanol and embedded in epoxy resin (PolyBed 812; Polyscience Inc). Ultrathin sections were cut out and stained with uranyl acetate and lead citrate before observation.
GTPS Binding Assays
Light-induced activation of bovine rod transducin by mouse rhodopsin and mouse green was measured by a GTPS binding assay as described previously (Terakita et al., 1998; Onishi et al., 2005). Rod outer segment (ROS) membranes containing rhodopsin or mouse green were prepared from retinas of dark-adapted wild-type or mG/mG mice by means of the sucrose floatation method (Shichida et al., 1987). The amount of visual pigments in ROS membranes was estimated by absorption spectroscopy after extraction of pigments with buffer E. Purification of transducin from bovine ROS was performed according to the methods described previously (Tachibanaki et al., 1997). The reaction was performed in 100 µl of Ringer solution (113 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 10 mM HEPES, 0.02 mM EDTA, pH 7.5), containing 30 nM (at 4°C) or 10 nM (at 37°C) pigment, 300 nM (at 4°C) or 100 nM (at 37°C) bovine rod transducin, 1 µM [35S]GTPS, and 1 µM GDP. The mixture was irradiated with a white light flash (SB80-DX; Nikon) that bleached 80% of visual pigments. After the mixture was incubated for a selected time in the dark, an aliquot (20 µl) was mixed with 250 µl of stop solution (20 mM Tris/Cl, 100 mM NaCl, 25 mM MgCl2, 1 µM GTPS, pH 7.4), and then immediately filtered through a nitrocellulose membrane to trap [35S]GTPS-bound transducin. The amount of light-dependent GTPS bound was estimated by subtracting the amount in the absence of the light stimulus from that in the presence of the light stimulus. The membrane was washed three times with 250 µl of wash solution (20 mM Tris/Cl, pH 7.4, 100 mM NaCl, 25 mM MgCl2) to remove free [35S]GTPS, and was air dried. The membrane was then put into 2 ml of aqueous counting scintillant (ACSII; GE Healthcare), and the amount of bound [35S]GTPS was measured using a liquid scintillation counter (LS600IC; Beckman Coulter).
Single-Cell Recordings
Mice kept in darkness for at least 12 h were killed by cervical dislocation and the eyes were removed and washed under dim red light conditions. All subsequent steps were performed under infrared light conditions using infrared image converters. The retina was isolated, cut into small pieces, and then finely chopped. Isolated pieces of retina were stored in Locke solution at 4°C before use. A suspension of cells was transferred to a recording chamber mounted on the microscope stage (TE300; Nikon). The solution in the recording chamber was perfused and heated to a temperature of 34–37°C, which was continuously monitored with a miniature thermocouple. The perfusion Locke solution, containing 112 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 10 mM HEPES, 20 mM NaHCO3, 3 mM Na2-succinate, 0.5 mM Na-glutamate, 0.02 mM EDTA, and 10 mM glucose at pH 7.4, was equilibrated with 95% O2/5% CO2. Individual cells were visualized under infrared light, using a CCD camera (ORCA-ER; Hamamatsu Photonics). Capillary glass was pulled and heat polished to fit the ROS diameter (1 µm), and filled with a solution, containing 140 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 3 mM HEPES, and 0.02 mM EDTA at pH 7.4. The bath solution was clamped by a bath clamp circuit (Baylor et al., 1984). A rod photoreceptor cell was drawn into the electrode to record the inward current of the outer segment. The circulating current was amplified by a current-to-voltage converter (Axopatch 200B; Axon Instruments), low-pass filtered by the Axopatch four-pole Bessel filter with a cutoff frequency of 1 kHz and by an eight-pole Bessel filter with a cutoff frequency of 30 Hz (900L8L; Frequency Devices), digitized at 400 Hz, and recorded with a pClamp 8.2 acquisition system (Axon Instruments) running on a personal computer. Unpolarized 20-ms light flashes were delivered from a 500-W xenon lamp (LX-500F; Sanso). Flashes at different wavelengths were obtained by passing the light beam through interference filters (max with the full bandwidth at half maximal transmittance as follows: 500 nm with 2.6 nm; 600nm with 6.9 nm; 652 nm with 9.0 nm; 676 nm with 9.7 nm) and neutral density filters (Toshiba). Unattenuated light was calibrated by a Digi-Ana Power Meter (PM245; Neoark) that was placed on the microscope stage at the same position as the recorded cells. As described previously (Baylor et al., 1979; Nikonov et al., 2005), the effective collecting area Ac() of the mouse rod for a flash of nm is given by
 | (1) |
(M–1 cm–1) is the extinction coefficient at nm of the pigment in solution, is the quantum efficiency of photoisomerization, C (M) is the concentration of the pigment in outer segment, and Vos (µm3) is the individual outer segment volume. The length of outer segments was calculated from digital images of the cells on the light microscope by image analysis software (AquaCosmos 2.5; Hamamastu Photonics) and its diameter was measured on the electron micrograph. The values of 500 are 40,200 for mouse rhodopsin (Imai et al., 2007) and 43,900 for mouse green, which was calculated based on 510 of mouse green (Onishi et al., 2005) and its absorption spectrum (Fig. 6 B, red trace). The quantum efficiency is = 0.67 for mouse rhodopsin (Dartnall, 1968) and = 0.61 for mouse green (see Results). The total number of rod photoreceptors in mouse retina was estimated as 4.1 x 106 from the product of the total area of the mouse retina (mm2) and the density of rods per unit area in the retina (rods mm–2); the total area of the mouse retina was estimated as 14.1 mm2 (Lyubarsky and Pugh, 1996) and the density of rods per unit area in the retina was estimated as 2.9 x 105 rods mm–2 provided that the ellipsoid region of inner segment with a diameter of 2.0 µm (Carter-Dawson and LaVail, 1979) was arranged with closest hexagonal packing columns. Since a diameter and length of outer segment was 1.4 and 15 µm for wild-type rod and 1.4 and 6.3 µm for mG/mG rod (TABLE I), Vos was 24 µm3 for wild type and 9.8 µm3 for mG/mG. Given that the number of rod photoreceptor cells contained in the retina was 4.1 x 106 cells for wild type and the number of cells was the same for the mG/mG retina, C was estimated to be 4.3 mM for wild-type rods and 1.2 mM for mG/mG rods. Thus, the effective collecting area Ac(500) (µm2) was 0.47 µm2 for wild-type rods and 0.054 µm2 for mG/mG rods, respectively.
Amplification Constant
The amplification constant was estimated by fitting the initial portion of the rising phase of the photocurrent response with Eq. 22 of Pugh and Lamb (1993):
 | (2) |
-nm light 
is given by
 | (3) |
nm and Ac() is the effective collecting area at nm given by Eq. 1. The responses evoked by 7–10 intensities for each cell were fit.
Spectral Sensitivities
Spectral sensitivities in single rod cells were determined by dividing the amplitude of the dim flash response (pA) by the given intensity (photons µm–2) at each wavelength. Each dim flash response was averaged from photoresponses elicited by more than 30 times identical flashes.
Noise Analysis
The one-sided power spectral densities were calculated using the Fast Fourier Transform weighted with a Hamming window (Clampfit 8.2 software; Axon Instruments). Power spectra were determined from 25–50 sweeps of 10.24-s-long segments, recorded from cells under each condition. Each current record was filtered at 30 Hz with an eight-pole Bessel low-pass filter and digitized at 400 Hz. Each spectral density was averaged and then smoothed by averaging over neighboring 3 points from 0 to 1 Hz, 5 points from 1 to 6 Hz, 10 points from 6 to 10 Hz, and 20 points above 10 Hz. For noise experiments in darkness, all light paths into the Faraday cage were sealed.
Instrumental Johnson noise level was calculated from the measured electrode resistance and absolute temperature using the Nyquist Equation, S(f) = 4kT/
, where k is Boltzmann's constant (1.38 x 1023 J/K), (
) is measured electrode resistance, and T (K) is absolute temperature. The measured power spectrum Sv(f) of the dim flash response as a function of frequency f was fit with the equation
 | (4) |
). For each cell, the value of the rate constant was determined by fitting the Independence expression with n = 4 stages to the dim flash response (Baylor et al., 1980). The rate of thermal activations of visual pigments in rods vd was then estimated as
 | (5) |
). The power spectrum of current fluctuation originated from components downstream from the visual pigments, Sc(f), were fit with the equation as follows
 | (6) |
).
In the bleaching experiment, the fractional bleaching pigments of the rod, F(Is, Ts), was estimated as follows
 |
We used the two-tailed unpaired Student's t test to determine the significance of difference among the results unless otherwise noted. Experimental data were fit using Levenberg–Marquardt algorithm (IgorPro5.0, WaveMetrics).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
20 kb in length, it seemed difficult to induce homologous recombination with the arm region of the rhodopsin gene. Therefore, we introduced mouse green cDNA (mG) that was followed by the rhodopsin polyadenylation signals (PA) and the neomycin-resistant marker (neo) into the first exon region of the rhodopsin gene. The targeting vector was electroporated into ES cell lines, and one of the 120 ES clones was identified as a homologous recombinant by selection with G418, a neomycin analogue, and Southern blot analysis. The knock-in mice were generated by blastocyst injection of the ES clones (Fig. 1 B), and it was confirmed that the knock-in mice were viable and fertile.
|
60% of that of rhodopsin (unpublished data). Western blot analysis with anti-rhodopsin monoclonal antibody (Rhodopsin 1D4) showed that rhodopsin was present in wild-type and heterozygous retinas (Fig. 1 D, top). The intensity of the rhodopsin band in the heterozygous lane was about half that in the wild-type lane, and the rhodopsin band was undetectable in the homozygous lane. On the other hand, in the wild-type lane, we could detect a faint band with anti-mouse green antibody, corresponding to mouse green endogenously expressed in cone cells, while in both Rh/mG and mG/mG lanes much denser bands were detected. (Fig. 1 D, bottom). These results indicated that the replacement of rhodopsin with mouse green was successful at the levels of both mRNA and protein.
Pigment Content in WT and mG/mG Rods.
Based on absorption spectroscopy, we estimated the amount of pigments in the retinas of 4-wk-old mice (Table I).
The amount of visual pigment (mouse green) per retina in mG/mG mice was 47 pmol, which was 11% of that (rhodopsin) in wild-type mice (410 pmol). On the assumption that each strain of mouse had the same number of rods in its retina, and using the number of 4.1 x 106 cells for wild type (see Materials and Methods), we estimated the number of visual pigments per rod to be 6.1 x 107 molecules in wild-type rods and 7.0 x 106 molecules in mG/mG rods. The photosensitivity at 500 nm of mouse green relative to that of rhodopsin was 0.99 (Fig. 2 A).
Thus, the quantum yield of mouse green was estimated to be 0.61 from the relationship that photosensitivity at 500 nm is proportional to the product of the extinction coefficient at 500 nm (500) and the quantum yield ().
|
|
; Johnson et al., 1993). Therefore, the heterozygous retinal extracts, which contain both rod and cone pigments, will undergo a biphasic change in the reaction with hydroxylamine. Fig. 2 B shows the time courses of hydroxylamine bleaching in the dark for rhodopsin (open squares), mouse green (red closed circles), and a mixture of rhodopsin and mouse green (open triangles) extracted from wild-type, mG/mG, and Rh/mG retinas, respectively. The time constants of bleaching reactions were 127 h for rhodopsin (open squares) and 1.93 h for mouse green (red closed circles). Based on the time constants of bleaching reactions for each type of visual pigments, the bleaching process of heterozygous retinas (open triangles) was fit with the double-exponential function with the time constants of rhodopsin (127 h) and mouse green (1.93 h). The ratio of the amount of rhodopsin to that of mouse green in Rh/mG retina was estimated to be 90 to 10. This ratio was comparable to the ratio of the amount of rhodopsin in wild-type retinas to the amount of mouse green in mG/mG retinas (9 to 1).
Content of Other Proteins of Transduction.
We next estimated the contents of phototransduction proteins other than the visual pigments in the retinas of 3-, 4-, and 6-wk-old mice (Fig. 2 C). The left panel of Fig. 2 C shows the blotting patterns of various transduction proteins in the retinas of 4-wk-old wild-type (Rh/Rh), Rh/mG, and mG/mG mice. The intensities of all bands in mG/mG retinas, especially that of the transducin -subunit, were faint relative to those in wild-type and Rh/mG retinas. It should be noted that age-dependent alteration was not observed at least during a period of 3–6 wk. The amounts of proteins in mG/mG retinas relative to those of wild-type retinas were quantified and the results are shown in the right panel of Fig. 2 C. The amount of transducin -subunit in mG/mG retinas was 0.2-fold of wild type, whereas those of the other proteins were 0.4–0.5-fold of wild type. From morphological studies, the total surface area of disc membrane or a surface area of plasma membrane of rod outer segment of mG/mG retinas was estimated to be 0.4-fold of wild type. Thus assuming that the same numbers of rod photoreceptor cells were present in wild-type and mG/mG retinas, the concentration, defined as the density per unit area of membrane, of transducin -subunit in mG/mG rod was estimated to be half of wild type, whereas the concentrations of other phototransduction proteins in mG/mG rod were similar to those in wild type. In Rh/mG retinas, the concentration of transducin was found to be 0.6-fold lower than that in wild-type retinas, but those of other proteins were similar to those in wild-type retinas.
Light Microscopy.
The retinal structure of 3-wk-old mice was first investigated by light microscopy. The structure appeared to be normal in both Rh/mG and mG/Mg mice (Fig. 3), although the length of the ROS in homozygous mice was about half that in wild type.
From careful measurements of cell dimension, we estimated that the outer segment length was 15 µm for wild-type rods but only 6.3 µm for mG/mG rods; i.e., 40% of wild type (Table I).
|
Electron Microscopy.
The structure of the ROS was further examined by electron microscopy, which showed that the outer segments appeared to be similar in structure between wild-type and Rh/mG mice at 3 wk of age (Fig. 3, M and N). Under careful observation, however, the discs in the ROS of mG/mG mice were well stacked and maintained normal size in the basal region but became gradually more disorganized toward the apical region with formation of vesicles (Fig. 3 O). The neural retina of mG/mG mice seemed to degenerate slowly at 3 wk of age, but in the retinas of 7-wk-old mice, the structure of the outer segments had severely degenerated (Fig. 3 P). It is likely that the level of expression of mouse green pigment was too low to maintain the stability of the outer segments disks. The same reasoning could be applied to the shortening of the ROS in 3-wk-old mG/mG mice. Judging from the morphological and biochemical observations described above, we decided to use 3-wk-old mice for electrophysiological experiments, to minimize the effect of degeneration.
Transducin Activation.
We examined transducin activations by mouse rhodopsin or mouse green expressed in ROS membrane. Fig. 4 shows that mouse green can activate transducin at 0°C with an efficiency similar to that of wild-type rhodopsin.
These results are in good agreement with previous reports (Fukada et al., 1989; Imai et al., 1997; Starace and Knox, 1997). We also measured the efficiency of transducin activation by mouse green at 37°C (Fig. 4, inset). Although the results showed that the initial rate of activation by mouse green was about half the rate of activation by wild-type rhodopsin, it appeared that the time resolution of the GTPS binding assay might not be sufficient for monitoring the rapid activation of transducin by cone visual pigments.
|
|
35-fold higher in mG/mG rods than in wild-type rods (Table I), comparable to the 32-fold difference estimated from electroretinogram experiments (unpublished data). This large difference in sensitivity stems in part from the different effective collecting area of the cells (as a result of the different pigment content), and when recalculated in terms of the numbers of photoisomerizations the difference is much smaller. Given the estimates of effective collecting areas at 500 nm, Ac(500), of 0.47 µm2 for wild-type and 0.054 µm2 for mG/mG rods (see Materials and Methods), we calculated the number of photoisomerizations required to half-saturate the response to be 9.7 R* for wild-type rods and 39 R* for mG/mG rods (Fig. 5 B and Table I); thus, when expressed in terms of photoisomerizations, the mG/mG rods are less sensitive by a factor of only 4.0.
Single-Photon Responses.
We stimulated rods with at least 30 consecutive identical dim flashes and estimated the amplitude of the single-photon response from the ensemble variance-to-mean ratio of the response amplitude based on the assumption of Poisson statistics (Baylor et al., 1979). The mean amplitude of the single-photon response from 13 mG/mG rods was 0.16 pA, which was 4.1-fold smaller than that from 17 wild-type rods (0.66 pA). The difference in amplitude of the single-photon response between wild-type and mG/mG rods should be compared with each other after expressing their single-photon responses as the fractional circulating currents. The amplitude of single-photon response in wild-type rods was 8.1% of the circulating current, while that in mG/mG rods was 2.7%. Thus the fractional circulating current in wild-type rods is threefold larger than that in mG/mG rods. Therefore, a large part of the 4.0-fold difference in the quantal sensitivity for 500-nm light (IoAc(500)) can be attributed to the difference in amplitude of the single-photon response. The integration time of dim flash responses for homozygous rods was 1.2-fold shorter than for wild-type rods, whereas the time-to-peak in mG/mG rods was similar to that in wild-type rods (Table I), suggesting that the shorter integration time results from an accelerated recovery phase. Since the time-to-peak of mG/mG rods was similar to that of wild-type rods, the lower sensitivity and the smaller amplitude of single-photon responses of mG/mG rods could be attributed to a reduction in the rising phase of the response, which related to the gain of the phototransduction cascade.
Amplification Constant.
The gain of phototransduction cascade can be evaluated as the amplification constant by fitting the rising phases of the photoresponses with Eq. 2. This equation is applicable only during the initial phase of the responses before inactivation reactions set in, and hence allows quantitative comparison of activation in each type of the cell. In the application of the amplification constants for the rising phase, it is assumed that the involvement of any inactivation step is neglected. It should be noted that we are unable to fully exclude the possibility that rapid intrinsic turn-off of the active state of mouse green occurs during the rising phase of the photoresponse (see Discussion).
The mean amplification constant from 13 wild-type rods was 25 s–2 (Fig. 5 C), which is relatively higher than those previously reported (13.6 s–2, Calvert et al., 2001; 8.3 s–2, Nikonov et al., 2006). The discrepancy might arise from the age-related variations such as the smaller cytoplasmic volume of the outer segment or higher concentrations of transduction components in young mice. The mean amplification constant from six mG/mG rods was 7.0 s–2 (Fig. 5 C), which is 3.6-fold smaller than that of wild-type rods.
In terms of the theory derived by Pugh and Lamb (1993), the difference in the amplification constant between that for control rods and that for rods containing mouse green can be explained by two factors: the outer segment volume and the rate of transduction activation by activated photopigment; however, this latter factor is likely to be affected by the concentration of transducin in the disc membranes. The amplification constant is predicted to be inversely proportional to the outer segment volume because a given number of activated molecules can change the cytoplasmic concentration of cyclic GMP more rapidly. Hence, our finding that the outer segment volume of mG/mG rods is only 42% that of wild-type rods would, in itself, predict an elevation of 2.4-fold in amplification constant. Hence the observation of an amplification constant that is lower by a factor of 3.6-fold in mG/mG rods would indicate that the rate of transducin activation is a factor of 8.6-fold lower in mG/mG rods. However, it would be expected that the 0.5-fold lower concentration of transducin in the membrane would contribute to this difference, so that the efficacy of the activated mouse green pigment molecule in activating transducin may only be a factor of 4.3-fold lower than the corresponding efficacy of activated rhodopsin.
Comparison of Photoresponses Evoked by E122Q Rhodopsin and Mouse Green in Single Rod Photoreceptor Cells
As mentioned above, comparison of the electrophysiological responses between wild-type and mG/mG rods showed that the amplitude of photoresponse evoked by mouse green was 3–4-fold lower than that evoked by rhodopsin. This value was derived both from single-photon responses and from sensitivity measurements after correction for the differences in morphology and in concentrations of visual pigment and transduction proteins between wild-type and mG/mG rods. Also, other factors, such as lower pigment concentrations in the disk membranes, may affect the amplification constant (Calvert et al., 2001). To exclude unavoidable uncertainty arising from such corrections, we attempted to evaluate photoresponses produced from rhodopsin and mouse green under identical conditions, using heterozygous mice whose rods contain both of these pigments. However, the spectral sensitivities of rhodopsin and mouse green (with max values at 502 and 510 nm, respectively), were so similar that it was difficult to selectively activate one type of these pigments. Therefore, we prepared a heterozygous (RhEQ/mG) mouse, with rods containing mouse green and E122Q rhodopsin. E122Q rhodopsin exhibits an absorption maximum (max = 487 nm) that is blue shifted 23 nm from the peak for mouse green but generates photoresponses similar to that of wild-type rhodopsin (Imai et al., 2007).
Pigment Content in RhEQ/mG Mice.
We estimated the ratio of the concentration of E122Q rhodopsin to that of mouse green present in heterozygous (RhEQ/mG) rods as 91 to 9, determined by the same method used for the Rh/mG mice (Fig. 6 A).
This ratio was almost the same as for wild-type rhodopsin and mouse green in Rh/mG rods (see above).
|
500, and the pigment concentration, C), we calculated the proportion of pigment photoactivated by 500-nm light in RhEQ/mG rods as 90% E122Q rhodopsin with the remainder being mouse green.
Other Wavelengths.
We then estimated the percentages of E122Q rhodopsin and mouse green photoactivated by 600-, 652-, and 676-nm lights in RhEQ/mG rods. Using single cell recordings, we measured the relative spectral sensitivities of E122Q rhodopsin from 10 RhEQ/RhEQ rods and those of mouse green from 11 mG/mG rods (Fig. 6 B and Table II).
Because we measured the spectral sensitivities by using the respective homozygous mice, these should be identical with the absorption spectra of E122Q rhodopsin and mouse green, respectively, if the quantum yields of these pigments are wavelength independent (Hurley et al., 1977), although we acknowledge that it is generally accepted that the quantum yields of visual pigments is slightly wavelength dependent. In fact, the spectral sensitivities were found to be superimposable on the absorption spectra of E122Q rhodopsin and mouse green, when the magnitudes were normalized at 500 nm (Fig. 6 B). The spectral sensitivities and absorption spectrum of E122Q rhodopsin were well fit with the spectral template (broken curve) reported by Govardovskii et al. (2000), whereas some deviation was apparent in the case of mouse green. This might be due to the difference in spectral shape between rhodopsin and long wavelength–sensitive cone visual pigments (Okano et al., 1989). From the ratio of each visual pigments photoactivated at 500 nm in RhEQ/mG rods and the relative spectral sensitivities of homozygous rods obtained in single cell recordings (Table II), the percentages of the pigments photoactivated in RhEQ/mG rods at the corresponding wavelengths could be determined. Thus, the percentages of E122Q rhodopsin photoactivated at 500, 600, 652, and 676 nm were estimated to be 90, 31, 16, and 12% of the total photoisomerizations, respectively (RhEQ:mG in Table III).
Importantly, the response evoked by 500-nm light is expected to be mainly from E122Q rhodopsin, and that evoked by the longer wavelengths is expected to be mostly from mouse green.
|
|
As previously described, the percentage of photoactivated E122Q rhodopsin was expected to be small when the rod was irradiated with long wavelength flashes. Interestingly, the ratio of the ensemble variance-to-mean decreased at longer wavelength (Table III; Fig. 6 C, inset). This was not due to any change in physiological state of the cells because the 500-nm photoresponses did not change through the experiment (c.f. the value of 500 nm pre and that of 500 nm post in Table III). The ensemble variance-to-mean ratio obtained in the present study does not represent the amplitude of the elementary response of either mouse green or E122Q rhodopsin because irradiation at any wavelength induces photoreactions of both mouse green and E122Q rhodopsin. However, these results strongly suggest that the amplitude of the elementary response generated by mouse green is significantly smaller than that generated by E122Q rhodopsin.
Calculation of the Elementary Response Elicited by Different Wavelengths.
To compare the amplitude of the elementary responses derived from mouse green and E122Q rhodopsin, we performed the following calculations. The amplitude of dim flash response, if it does not exceed 20% of the maximal response, is proportional to the flash intensity (Field and Rieke, 2002). Therefore, the flash sensitivity of heterozygous (RhEQ/mG) rods at 0 nm, 
(0), is obtained by the following equation:
 | (7) |
EQ and mG represent the mean number of photoisomerizations of E122Q rhodopsin and mouse green in a RhEQ/mG rod evoked by a dim flash of intensity i at 0 nm, respectively. 
(0) and 
(0) represent the effective collecting area at 0 nm in RhEQ/mG rods for E122Q rhodopsin and that for mouse green, respectively. gEQ and gmG are the peak amplitude of the elementary responses produced by a single photoisomerization of E122Q rhodopsin and mouse green, respectively.
In a similar way, the sensitivity of RhEQ/mG rods at 1 nm, (1), is represented as follows:
 | (8) |
(1) and (1) represent the effective collecting areas at 1 nm in RhEQ/mG rods of E122Q rhodopsin and mouse green, respectively.
From Eqs. 1, 7, and 8, the ratio of the amplitude of the elementary response of mouse green relative to that of E122Q rhodopsin is derived as follows:
 | (9) |
and 
are the extinction coefficient at 1 nm relative to that at 0 nm of E122Q rhodopsin and mouse green, respectively. 
represents the sensitivity of RhEQ/mG rods at 1 nm relative to that at 0 nm. 
is the ratio of photosensitivity at 0 
