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• W2058709834 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. 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. Light absorption by rhodopsin in rod photoreceptor cells results in the activation of a G protein-mediated signal transduction cascade that eventually generates an electrical response (1Yau K.W. Investig. Ophthalmol. Vis. Sci. 1994; 35: 9-32PubMed Google Scholar). The key proteins in this cascade have been identified, and their molecular properties as well as interactions with each other have been extensively investigated (2Helmreich E.J. Hofmann K.P. Biochim. Biophys. Acta. 1996; 1286: 285-322Crossref PubMed Scopus (127) Google Scholar, 3Shichida Y. Imai H. Cell Mol. Life Sci. 1998; 54: 1299-1315Crossref PubMed Scopus (212) Google Scholar). A current question is how well these properties and interactions correlate with the response profile of the photoreceptor cells. Although the gene knock-out approach has been very useful in addressing this question for the proteins rhodopsin kinase and arrestin (4Xu J. Dodd R.L. Makino C.L. Simon M.I. Baylor D.A. Chen J. Nature. 1997; 389: 505-509Crossref PubMed Scopus (271) Google Scholar, 5Chen C.K. Burns M.E. Spencer M. Niemi G.A. Chen J. Hurley J.B. Baylor D.A. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3718-3722Crossref PubMed Scopus (279) Google Scholar), this strategy is less appropriate for rhodopsin and G protein, because the deletions of these proteins eliminated the light response (6Lem J. Krasnoperova N.V. Calvert P.D. Kosaras B. Cameron D.A. Nicolo M. Makino C.L. Sidman R.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 736-741Crossref PubMed Scopus (327) Google Scholar, 7Calvert P.D. Krasnoperova N.V. Lyubarsky A.L. Isayama T. Nicolo M. Kosaras B. Wong G. Gannon K.S. Margolskee R.F. Sidman R.L. Pugh Jr., E.N. Makino C.L. Lem J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13913-13918Crossref PubMed Scopus (290) Google Scholar). The gene knock-in approach is an alternative way, with a mutant protein replacing the wild-type (WT) 8The abbreviations used are: WT, wild type; ROS, rod outer segment.8The abbreviations used are: WT, wild type; ROS, rod outer segment. version. The maintenance of the same expression level of the protein in this procedure is important, because the interpretation can be difficult otherwise. For example, the photoresponse profile is altered when the rhodopsin content in rods is halved (8Calvert P.D. Govardovskii V.I. Krasnoperova N. Anderson R.E. Lem J. Makino C.L. Nature. 2001; 411: 90-94Crossref PubMed Scopus (146) Google Scholar, 9Liang Y. Fotiadis D. Maeda T. Maeda A. Modzelewska A. Filipek S. Saperstein D.A. Engel A. Palczewski K. J. Biol. Chem. 2004; 279: 48189-48196Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Our past work on comparing rhodopsin and cone pigments (10Shichida Y. Imai H. Imamoto Y. Fukada Y. Yoshizawa T. Biochemistry. 1994; 33: 9040-9044Crossref PubMed Scopus (72) Google Scholar) has shown that their photosensitivities are not so different, but the meta-II (the G protein-activating state), as well as the subsequent meta-III, intermediates of cone pigments exhibit faster decay than those of rhodopsin. In addition, the equilibrium between meta-II and its precursor, meta-I, is different between rhodopsin and cone pigments (11Imai H. Kuwayama S. Onishi A. Morizumi T. Chisaka O. Shichida Y. Photochem. Photobiol. Sci. 2005; 4: 667-674Crossref PubMed Scopus (29) Google Scholar). Finally, we have found that the amino acid residues at positions 122 and 189 underlie these differences in molecular properties (12Imai H. Kojima D. Oura T. Tachibanaki S. Terakita A. Shichida Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2322-2326Crossref PubMed Scopus (119) Google Scholar, 13Kuwayama S. Imai H. Hirano T. Terakita A. Shichida Y. Biochemistry. 2002; 41: 15245-15252Crossref PubMed Scopus (49) Google Scholar). Thus, to obtain insight into how the molecular properties of rhodopsin correlate with the photoresponse profile of rod cells, and to elucidate the differences in response properties between rods and cones, we have generated a knock-in mouse line in which E122Q rhodopsin replaced WT rhodopsin in the rod cells. Mutation of the Glu122 residue in chicken rhodopsin into the corresponding residue in cone pigments results in a faster decay of meta-II, a shift of the meta-I/meta-II equilibrium toward meta-I (12Imai H. Kojima D. Oura T. Tachibanaki S. Terakita A. Shichida Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2322-2326Crossref PubMed Scopus (119) Google Scholar, 14Nathans J. Biochemistry. 1990; 29: 937-942Crossref PubMed Scopus (177) Google Scholar), and a faster regeneration of the pigment (15Imai H. Terakita A. Tachibanaki S. Imamoto Y. Yoshizawa T. Shichida Y. Biochemistry. 1997; 36: 12773-12779Crossref PubMed Scopus (61) Google Scholar). Thus, this point mutant recapitulates certain cone-pigment-like properties in rhodopsin. Residue 122 is located near the β-ionone ring of the chromophore in the helical core of the visual pigment (16Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4971) Google Scholar), and its substitution has little effect on the interaction of the pigment with other proteins, such as G protein. More importantly, the replacement of only one codon in the native rhodopsin gene without disturbing the exon-intron arrangement should give similar transcription and translation as WT. This knock-in mouse allows us to compare the molecular properties of WT and E122Q rhodopsin in the same native environment, i.e. membranes of the rod outer segment (ROS). Our findings reported here clearly show that several of the rhodopsin molecular properties, such as absorption spectrum, photosensitivity, shift in the meta-I/meta-II equilibrium, and meta-III decay, do correlate well with the rodresponse profile and the recovery of rod sensitivity after a bleaching light. The meta-II decay also correlates well with the decline phase of the rod response when arrestin is absent. Generation of Rhodopsin-mutant Mouse—Mutant mice with E122Q rhodopsin were generated as previously reported (17Chisaka O. Capecchi M.R. Nature. 1991; 350: 473-479Crossref PubMed Scopus (672) Google Scholar, 18Onishi A. Hasegawa J. Imai H. Chisaka O. Ueda Y. Honda Y. Tachibana M. Shichida Y. Zoolog Sci. 2005; 22: 1145-1156Crossref PubMed Scopus (13) Google Scholar). Briefly, the mouse rhodopsin genomic clones were modified by replacing Glu122 (GAA) with Gln (GAG) in the second exon and introducing the positive selection marker (PGKneopA) into the first intron in the reverse direction (Fig. 1A). The E122Q-positive ES cells were screened from electroporated R1 ES cells derived from the 129/Sv x 129Sv-CP mouse line, and chimeric mice were generated by injecting them into C57BL/6J blastocysts, which were subsequently implanted into the uteri of pseudo-pregnant foster females. The chimeras were mated with C57BL/6J mice to generate heterozygous mice that were then inbred to generate homozygous mice. The targeted allele has been maintained in both 129/SvJ and C57BL/6J backgrounds according to the guidelines for animal experiments at Kyoto University. Estimation of Protein Expression Level—Protein expression levels were estimated by spectroscopy and Western blotting. Retinas were isolated from mouse eyes, homogenized, and extracted by buffer A (1% dodecylmaltoside, 50 mm HEPES, 140 mm NaCl, 3 mm MgCl2, pH 6.5) for spectroscopy or by SDS-PAGE sample buffer (8% SDS, 0.125 m Tris/Cl (pH 6.8), 20% glycerol, 5% mercaptoethanol) for Western blotting. The concentration of pigment in the extract solubilized by buffer A was estimated from the maximum absorbance in the difference spectrum before and after irradiation with >480 nm light in the presence of 100 mm hydroxylamine. For calculations, we used molar extinction coefficients of both rhodopsin and E122Q rhodopsin. Molar extinction coefficients of rhodopsin and E122Q rhodopsin at their absorption maxima (502 and 487 nm, respectively) are 40,200 ± 2,100 and 44,800 ± 2,200 m-1 cm-1, respectively, 9H. Imai, unpublished results. which were estimated using a method previously described (19Okano T. Fukada Y. Shichida Y. Yoshizawa T. Photochem. Photobiol. 1992; 56: 995-1001Crossref PubMed Scopus (48) Google Scholar) and the acid-denaturing method (20Sakamoto T. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 249-253Crossref PubMed Scopus (34) Google Scholar). These values are in good agreement with those estimated previously for bovine rhodopsin (21Sakmar T.P. Franke R.R. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8309-8313Crossref PubMed Scopus (596) Google Scholar, 22Nakayama T.A. Khorana H.G. J. Biol. Chem. 1991; 266: 4269-4275Abstract Full Text PDF PubMed Google Scholar). The expression levels for WT and E122Q rhodopsins, transducin, and rhodopsin kinase were also estimated by Western blotting with antibodies against rhodopsin (1D4), transducin (23Suzuki T. Narita K. Yoshihara K. Nagai K. Kito Y. Zoolog. Sci. 1993; 10: 425-430PubMed Google Scholar), and rhodopsin kinase (sc-561, Santa Cruz). Histology—Eyes were removed and fixed for 4 h at 4 °C in 4% paraformaldehyde in phosphate-buffered saline. After cryoprotection in cold 30% sucrose, the tissue was mounted with Tissue-Tek OCT and sectioned at 10-μm thickness along the vertical meridian passing through the optic nerve. Staining was with 1% toluidine blue O. Spectroscopy of Pigments in Native ROS Membranes—ROS membranes were isolated by a conventional sucrose flotation method (24Shichida Y. Ono T. Yoshizawa T. Matsumoto H. Asato A.E. Zingoni J.P. Liu R.S. Biochemistry. 1987; 26: 4422-4428Crossref PubMed Scopus (48) Google Scholar) from retinas of mice dark-adapted for 12 h. The membrane fractions were suspended in buffer B (112.5 mm NaCl, 3.6 mm KCl, 2.4 mm MgCl2, 1.2 mm CaCl2, 10 mm HEPES (pH 7.4)) and sonicated. Absorption spectroscopy was performed as previously reported on a Shimadzu MPS-2000 or UV-2400 spectrophotometer (25Shichida Y. Tachibanaki S. Mizukami T. Imai H. Terakita A. Methods Enzymol. 2000; 315: 347-363Crossref PubMed Google Scholar), and a specially constructed CCD spectrophotometer (26Morizumi T. Imai H. Shichida Y. Biochemistry. 2005; 44: 9936-9943Crossref PubMed Scopus (26) Google Scholar). The sample temperature was regulated by thermostat to within 0.1 °C in the cell holder of the spectrophotometer. Light sources for sample irradiation were a 1-kW tungsten halogen lamp (Rikagaku Seiki) and a short arc power flash (Nissin Electronic Co., pulse duration = 350 μs), which were attached in the Shimadzu spectrophotometer and the CCD spectrophotometer, respectively. A glass cut-off (VY50, VY52, or VY54; Toshiba) or an interference (500 nm; Nihon Shinku) filter was used for selection of the irradiation wavelengths. Details of procedures for specific measurements including irradiation conditions are described in the figure legends. Single-cell Recordings—WT and homozygous E122Q mice were housed in a 12/12-h light-dark cycle. Mice lacking arrestin (Arr-/-) (4Xu J. Dodd R.L. Makino C.L. Simon M.I. Baylor D.A. Chen J. Nature. 1997; 389: 505-509Crossref PubMed Scopus (271) Google Scholar), including E122Q, Arr-/- mice, were reared in constant darkness. Animals raised in a light-dark cycle were dark-adapted overnight before the experiment. Tissue preparation and recording techniques followed procedures described elsewhere (27Burns M.E. Mendez A. Chen J. Baylor D.A. Neuron. 2002; 36: 81-91Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Briefly, an animal was euthanized by CO2 asphyxiation/cervical dislocation and the eyes were removed under dim red light. All further manipulations were performed under infrared light. The retinas removed from the eyes were chopped into small pieces with a razor blade. Small pieces of the retina were placed in the experimental chamber on the stage of an inverted microscope and perfused with bicarbonate-buffered solution (112.5 mm NaCl, 3.6 mm KCl, 2.4 mm MgCl2, 1.2 mm CaCl2, 10 mm HEPES (pH 7.4), 20 mm NaHCO3, 3 mm sodium succinate, 0.5 mm sodium glutamate, 0.02 mm EDTA, and 10 mm glucose). The solution was bubbled with 95% O2, 5% CO2, and warmed to 36-38 °C in a flow heater (28Matthews H.R. J. Physiol. 1999; 518P: 13PGoogle Scholar) before it entered the experimental chamber. Membrane current was recorded with a suction electrode from an ROS projecting from a piece of retina. The recording electrode was filled with 140 mm NaCl, 3.6 mm KCl, 2.4 mm MgCl2, 1.2 mm CaCl2, 3 mm HEPES (pH 7.4), 0.02 mm EDTA, and 10 mm glucose. 20-ms flashes were delivered from a calibrated light source via computer-controlled shutters. Light intensity and wavelength were changed by using calibrated neutral density and interference filters. The current was amplified, low-pass filtered at 30 Hz, digitized at 1 kHz, and stored on a computer for subsequent analysis. Simulation of Rod Photoresponses—The rod-response profile was simulated by the model described previously (29Ichikawa K. Neurosci. Res. 1994; 19: 201-212Crossref PubMed Scopus (6) Google Scholar) with some modifications. First, the reaction scheme was modified so that transducin can bind to phosphodiesterase at the mole ratio of 1:1. Second, parameters such as rod outer segment morphology (30Carter-Dawson L.D. LaVail M.M. J. Comp. Neurol. 1979; 188: 245-262Crossref PubMed Scopus (527) Google Scholar) and phosphodiesterase deactivation rate constant (31Makino C.L. Dodd R.L. Chen J. Burns M.E. Roca A. Simon M.I. Baylor D.A. J. Gen. Physiol. 2004; 123: 729-741Crossref PubMed Scopus (133) Google Scholar) appropriate for mouse rods were substituted for those for amphibian rods used in the previous model. The reaction model was constructed with the A-cell 5.1 software (32Ichikawa K. Bioinformatics. 2001; 17: 483-484Crossref PubMed Scopus (27) Google Scholar, 33Ichikawa K. Neuroinformatics. 2005; 3: 49-64Crossref PubMed Google Scholar). Generation of E122Q Knock-in Mice—WT rhodopsin was replaced by its E122Q mutant in mouse by using gene targeting (Fig. 1, A and B). The homozygous animals were viable and fertile. The expression level of E122Q rhodopsin in these animals was indistinguishable from that of WT rhodopsin (Fig. 1C), and the levels of transducin, rhodopsin kinase, and green-sensitive cone opsin were also indistinguishable (data not shown). The homozygous animals with the positive selection marker (PGKneopA) still in the genome had an expression level of visual pigment about 72% of that of WT animals (Fig. 1C) but their overall retinal structure, especially that of ROS, appeared to be normal under light microscopy (Fig. 1D). The numbers of photoreceptor nuclei in WT and mutant mice were almost identical. That is, rows of photoreceptor nuclei in WT and mutant mice were 10.8 ± 1.1 and 10.2 ± 0.9, respectively, at 10 weeks of age (n = 20 from 3 animals for each genotype). The lengths of outer segment were also indistinguishable between WT (18 ± 0.8 μm) and E122Q mutants (19 ± 1.6 μm). Molecular Properties of WT and E122Q Rhodopsins—We first asked whether the E122Q mutant rhodopsin in mouse ROS exhibited molecular properties different from those of WT, as might be expected from our previous heterologous expression experiments with the same mutation in chicken rhodopsin (12Imai H. Kojima D. Oura T. Tachibanaki S. Terakita A. Shichida Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2322-2326Crossref PubMed Scopus (119) Google Scholar). The results are summarized in Fig. 2 and Table 1. The absorption maximum of E122Q rhodopsin extracted from ROS with buffer A was about 15 nm blue-shifted from that of the corresponding WT rhodopsin (Fig. 2A), similar to the findings with bovine (14Nathans J. Biochemistry. 1990; 29: 937-942Crossref PubMed Scopus (177) Google Scholar, 21Sakmar T.P. Franke R.R. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8309-8313Crossref PubMed Scopus (596) Google Scholar, 22Nakayama T.A. Khorana H.G. J. Biol. Chem. 1991; 266: 4269-4275Abstract Full Text PDF PubMed Google Scholar, 34Zhukovsky E.A. Oprian D.D. Science. 1989; 246: 928-930Crossref PubMed Scopus (426) Google Scholar, 35Nagata T. Terakita A. Kandori H. Shichida Y. Maeda A. Biochemistry. 1998; 37: 17216-17222Crossref PubMed Scopus (54) Google Scholar), chicken (15Imai H. Terakita A. Tachibanaki S. Imamoto Y. Yoshizawa T. Shichida Y. Biochemistry. 1997; 36: 12773-12779Crossref PubMed Scopus (61) Google Scholar), and mouse (11Imai H. Kuwayama S. Onishi A. Morizumi T. Chisaka O. Shichida Y. Photochem. Photobiol. Sci. 2005; 4: 667-674Crossref PubMed Scopus (29) Google Scholar) E122Q rhodopsins expressed in culture cells. The photosensitivity of E122Q rhodopsin at 500 nm was 0.92 ± 0.01 of WT (Fig. 2B, Table 1). Whereas the molecular extinction coefficient at λmax was higher for E122Q rhodopsin compared with WT rhodopsin, at 500 nm the two pigments had very similar extinction coefficients (Fig. 2A and see “Experimental Procedures”). Thus, the smaller photosensitivity of E122Q rhodopsin at 500 nm indicates that E122Q rhodopsin had a quantum yield lower than that of WT rhodopsin. We next investigated the thermal equilibrium between meta-I and meta-II after irradiating WT and E122Q rhodopsins in ROS membranes. We found that the equilibrium was significantly shifted toward meta-I in E122Q rhodopsin compared with WT (Fig. 2, C and D). Under the conditions described in the legends of Fig. 2, C and D, the ratio of meta-I to meta-II in E122Q rhodopsin was 34:66, while that in WT was 18:82. These values were estimated by fitting the respective spectrum obtained at 100 ms after 500 nm irradiation of each pigment with a linear combination of the spectra for meta-I and meta-II obtained independently (26Morizumi T. Imai H. Shichida Y. Biochemistry. 2005; 44: 9936-9943Crossref PubMed Scopus (26) Google Scholar). Within a few seconds after irradiation, E122Q meta-II decayed with a time constant about 10 times faster than that of WT meta-II (Fig. 2, E and F). The decay of E122Q meta-III was also 2.4-fold as fast as WT meta-III (Fig. 2, G and H, see also Fig. 5C).TABLE 1Parameters for the molecular properties of mouse rhodopsinWild typeE122Qλmax (nm)aProteins were detergent-extracted from dark-adapted eyes and measured at 2 °C.502 ± 1.3 (7)487 ± 1.4 (7)Rel. photosensitivity at 500 nmaProteins were detergent-extracted from dark-adapted eyes and measured at 2 °C.1.00.92 ± 0.01 (5)Meta-II decay time constants (s)bProteins remained in ROS membranes that were suspended in buffer B (pH 7.4) at 37 °C.95 ± 15 (7)7.7 ± 3.5 (8)Meta-III decay time constants (min)bProteins remained in ROS membranes that were suspended in buffer B (pH 7.4) at 37 °C.17.7 ± 3.0 (3)7.4 ± 3.7 (3)Regeneration time constants177 ± 32 (3)47 ± 10 (3)From opsin + 11-cis-retinal (s)cProteins remained in ROS membranes that were suspended in buffer B (pH 7.4) at 2 °C.a Proteins were detergent-extracted from dark-adapted eyes and measured at 2 °C.b Proteins remained in ROS membranes that were suspended in buffer B (pH 7.4) at 37 °C.c Proteins remained in ROS membranes that were suspended in buffer B (pH 7.4) at 2 °C. Open table in a new tab FIGURE 5Comparison 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 (λmax 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 460 nm 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Photoresponses of WT and E122Q Mice—We recorded with a suction pipette the photoresponse from individual rods protruding from fragments of retina in the absence of pigment epithelium (see “Experimental Procedures”). The maximum flash response amplitudes of WT and E122Q rods were similar. The action spectra of WT and E122Q rods (Fig. 3C) were best fit by modified A1 visual-pigment templates (36Govardovskii V.I. Calvert P.D. Arshavsky V.Y. J. Gen. Physiol. 2000; 116: 791-794Crossref PubMed Scopus (20) Google Scholar) with λmax = 496 ± 3 nm (n = 10) for WT and 480 ± 3 nm (n = 6) for E122Q rods, in good agreement with the absorption maxima of the corresponding rhodopsins described earlier (Fig. 2A). At 500 nm, the flash sensitivity, defined as the reciprocal of half-maximal flash strength (1/I0), was lower for E122Q rods, being 0.72 that of WT (Table 2). To derive the single-photon response amplitude, we stimulated a cell with a series of 30-100 identical dim flashes. According to Poisson statistics, the amplitude of the single-photon response can be estimated from the ensemble variance to mean ratio of the response amplitude. The single-photon response amplitude (SØ) derived in this way was 0.45 ± 0.03 pA (n = 26) for mutant rods versus 0.56 ± 0.04 pA (n = 16) for WT rods (Fig. 3D and Table 2). The rate of activation of the single-photon response of the E122Q mutant rod is slightly smaller than that of WT. The decline phase of the dimflash response was notably slower in mutant rods, as reflected by the longer integration time (Fig. 3D and Table 2). These results would be due to the altered molecular properties of rhodopsin by the mutation of E122Q. Thus we tried to simulate the rod response by altering the molecular properties of rhodopsin.TABLE 2Parameters for the single-cell response of mouse rodsWild typeE122QIdaId, dark current. (pA)10.8 ± 0.4 (23)9.8 ± 0.4 (19)IobIo, flash strength at 500 nm that gives the half-maximal response. (photons μm−2)33.9 ± 1.6 (23)46.6 ± 1.6 (19)cDifference from WT being statistically significant, p < 0.005.SφdSφ, single-photon response. (pA)0.56 ± 0.04 (16)0.45 ± 0.03 (26)eDifference from WT being statistically significant, p < 0.05.tpftp, time to peak. (ms)160 ± 3 (16)175 ± 3 (26)cDifference from WT being statistically significant, p < 0.005.tigti, integration time defined as the area of the dim flash response divided by its peak amplitude. (ms)309 ± 16 (16)379 ± 22 (26)eDifference from WT being statistically significant, p < 0.05.a Id, dark current.b Io, flash strength at 500 nm that gives the half-maximal response.c Difference from WT being statistically significant, p < 0.005.d Sφ, single-photon response.e Difference from WT being statistically significant, p < 0.05.f tp, time to peak.g ti, integration time defined as the area of the dim flash response divided by its peak amplitude. Open table in a new tab Simulation of Photoresponse by Chemical Reaction Model—Can we explain the differences in response characteristics between WT and E122Q rods by the differences between WT and E122Q rhodopsins? E122Q rods exhibited a flash sensitivity at 500 nm about 0.7 times that of WT. This difference can be accounted for by the combined difference in pigment sensitivity at 500 nm (0.9) and difference in the magnitude of the singlephoton response (0.8) (0.9 × 0.8 = 0.72). Interestingly, because the size of the single-photon response in E122Q mutant was about 0.8 times that of wild-type, whereas the integration time recorded in E122Q mutant was longer (about 1.3 times), the total area under the response (amplitude × time) was quite similar in both cases. Simulation analysis based on a model previously described (29Ichikawa K. Neurosci. Res. 1994; 19: 201-212Crossref PubMed Scopus (6) Google Scholar) clearly showed that these differences in the single-photon response between E122Q and WT rods could be accounted for by a change in the meta-I/meta-II equilibrium. In addition, the shift in the meta-I to meta-II equilibrium used in our simulation correctly predicted the small difference in the rates of activation of the single photon response between WT and E122Q rods (Fig. 3D). The simulation was able to reproduce these observed differences in amplitude and kinetics when meta-II was assumed to be the only intermediate that can be phosphorylated by rhodopsin kinase (Fig. 4, main panel at bottom), but not when only meta-I is phosphorylated or when both meta-I and II can be phosphorylated (Fig. 4, insets A and B at bottom). Indeed, it can be seen that the time to peak and the response recovery are delayed in the E122Q mutant when only meta-II is phosphorylated. Otherwise, they are unchanged when both intermediates are phosphorylated, a" @default.
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• W2058709834 cites W2041441066 @default.
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