|Molecular Vision 2001;
Received 8 February 2001 | Accepted 19 March 2001 | Published 20 March 2001
RGS9-1 is required for normal inactivation of mouse cone phototransduction
A. L. Lyubarsky,1 C.-K. Chen,2 F. Naarendorp,3 X. Zhang,4
M. I. Simon,5
E. N. Pugh, Jr.1
1F. M. Kirby Center and Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA; 2Department of Ophthalmology and Human Genetics, University of Utah, Salt Lake City, UT; 3Department of Psychology, Northeastern University, Boston, MA; 4Department of Biochemistry, Baylor College of Medicine, Houston, TX; 5Division of Biology, California Institute of Technology, Pasadena, CA
Correspondence to: E. N. Pugh, Jr., Ph.D., F. M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Stellar-Chance Building, Room 309B, 422 Curie Boulevard, Philadelphia, PA, 19104-6069; Phone: (215) 898-2403; FAX: (215) 898-7155; email: firstname.lastname@example.org
Purpose: To test the hypothesis that Regulator of G-protein Signaling 9 (RGS9-1) is necessary for the normal inactivation of retinal cones.
Methods: Mice having the gene RGS9-1 inactivated in both alleles (RGS9-1 -/-) were tested between the ages 8-10 weeks with electroretinographic (ERG) protocols that isolate cone-driven responses. Immunohistochemistry was performed with a primary antibody against RGS9-1 (anti-RGS9-1c), with the secondary conjugated to fluorescein isothiocyanate, and with rhodamine-conjugated peanut agglutinin.
Results: (1) Immunohistochemistry showed RGS9-1 to be strongly expressed in the cones of wildtype (WT is C57BL/6) mice, but absent from the cones of RGS9-1 mice. (2) Cone-driven b-wave responses of dark-adapted RGS9-1 -/- mice had saturating amplitudes and sensitivities in the midwave and UV regions of the spectrum equal to or slightly greater than those of WT (C57BL/6) mice. (3) Cone-driven b-wave and a-wave responses of RGS9-1 -/- mice recovered much more slowly than those of WT after a strong conditioning flash: for a flash estimated to isomerize 1.2% of the M-cone pigment and 0.9% of the UV-cone pigment, recovery of 50% saturating amplitude was approximately 60-fold slower than in WT.
Conclusions: (1) The amplitudes and sensitivities of the cone-driven responses indicate that cones and cone-driven neurons in RGS9-1 -/- mice have normal generator currents. (2) The greatly retarded recovery of cone-driven responses of RGS9-1 -/- mice relative to those of WT mice establishes that RGS9-1 is required for normal inactivation of the cone phototransduction cascades of both UV- and M-cones.
In the phototransduction G-protein-coupled receptor (GPCR) cascades of vertebrate rods and cones, signal amplification is effected primarily by two enzymes: (1) photoactivated visual pigment (R*), which catalytically generates the activated form of the G-protein (G* = Gta-GTP), and (2) the activated complex (G*-E*) of the effector enzyme, phosphodiesterase (E) with G* . The timely inactivation of these two enzymatic amplifiers after their activation by a light stimulus is necessary for normal visual function, and proteins specialized to inactivate each of them have been found and characterized in rods. Thus, in rods, rhodopsin kinase (GRK1) and arrestin (Arr1) are both necessary for normal inactivation of R*, and null defects in these latter proteins lead to Oguchi's disease, a form of stationary night blindness in which long-lived decay products of R* cause rods to recover extremely slowly from light exposure [2,3]. Recently, it has been found that normal inactivation of the G*-E* complex in rods requires a GTPase-activating protein or "GAP", RGS9-1, which is expressed only in the retina [4,5]. RGS9-1 has an obligatory requirement for a specific G-protein b-subunit, Gb5-L, to be stably expressed and present in rod outer segments, where the complex, RGS9-1/ Gb5-L, performs its GAP function [6,7]. The rods of mice null for RGS9-1 have extremely slowed photoresponses, recovering from moderate intensity flashes with a dominant time constant (9 s) that is 45-fold longer than that in WT (ca. 0.2 s) , and thus these animals have a "stationary night blindness" originating in the slowed inactivation of the second cascade amplifier.
Relatively little is known about the mechanisms of inactivation of cone phototransduction cascades, as compared with what is known about the mechanisms of inactivation in rods. Because the primary proteins of the cascades of cones, the photopigment GPCR, the G-protein, the PDE, and the cyclic nucleotide-gated channel, all are distinct isoforms from those of rods, it might be expected that the inactivating proteins of cones would also be distinct from those in rods. However, recent work has established that the normal inactivation of R* in murine cones requires GRK1, "rhodopsin kinase", violating this expectation , and also that bovine cones strongly express RGS9-1 . Here we test the hypothesis that the GAP factor, RGS9-1 is necessary for normal inactivation of cones in the mouse by examining the cone-driven ERG responses of mice homozygously null for this protein.
Animals and husbandry
All experimental procedures were done in compliance with NIH guidelines, as approved by the respective Institutional Animal Care and Use Committees of the University of Pennsylvania, the California Institute of Technology, and Baylor College of Medicine. RGS9-1 -/- mice were derived at the California Institute of Technology on C57BL/6-129/SvJ background as described elsewhere . C57BL/6 mice were used as controls, and hereafter are referred to as "WT." All animals employed for ERG recordings were born and maintained under controlled ambient illumination on 12/12 light/dark cycle with the illumination level at 2.5 photopic lux as described previously [11,12].
Whole eyes were removed from euthanized WT and RGS9-1 -/- mice and fixed in 4% paraformaldehyde-phosphate buffered saline (PBS, pH 7.2) for 10 to 16 h at 4 °C. After fixing, the eyes were placed in 30% sucrose (w/v)-PBS for 1 h at 4 °C, then embedded in OCT compound Tissue-Tek (Sakura Finetek, Inc., Torrance, CA). Frozen sections were cut 14 mm thick, thawed at room temperature for 2 min, post-fixed in 1:1 methanol:acetone (vol/vol) for 10 min, and thereafter processed at room temperature. They were rehydrated in PBS (pH 7.2) for 20 min and blocked with 10% sheep serum (Sigma Chemical, St. Louis, MO) PBS for 1 h. To stain RGS9, tissue sections were incubated with primary antibody to RGS9, anti-RGS9-1c , at a 1:200 dilution in 10% sheep serum-PBS. To stain Gb5, a polyclonal antibody directed against a peptide epitope  common to Gb5-L and Gb5-S was used, also at a 1:200 dilution in 10% sheep serum-PBS. All sections were incubated with primary antibody overnight in a humidified atmosphere. After being washed 3 x 5 min in PBS, sections were incubated for 1 h with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin G (Vector, Burlingame, CA), at a 1:25 dilution, and, when included, rhodamine-conjugated peanut agglutinin (Vector) at a 1: 100 dilution in 10% sheep serum-PBS in a humidified atmosphere. Peanut agglutin has been established to bind specifically to cone photoreceptor cells . Sections were washed 3 x 10 min in PBS and mounted in aqueous mounting medium (Gel/Mount; Biomeda, Foster City, CA). Sections were examined and photographed with a confocal fluorescence microscope (Zeiss LSM 510) and a Spot digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI), and processed with Adobe Photoshop (v. 5.5).
Electroretinographic (ERG) recordings were made when animals were between 8 and 12 weeks of age. Protocols for ERG recordings, stimulus intensity calibration and computation of the amount of visual pigments isomerized by the flashes, and methods of isolating cone-driven responses are described in detail elsewhere [9,12]. In brief, ERGs were recorded from anesthetized mice with a differential amplifier with bandwidth 0.1 Hz to 1 KHz, and sampled and digitized at 5 KHz. The corneal electrode was a platinum wire, while the reference electrode was a tungsten needle inserted subcutaneously in the forehead. The recording chamber served dually as a Faraday cage and a ganzfeld, with ports and baffles for illumination. Cone-signal isolation was effected with steady backgrounds estimated to produce 6000 photoisomerizations rod-1s-1 in WT mice, and 3000 photoisomerizations rod-1s-1 in RGS9-1 -/- mice.
Cones of WT mice strongly express RGS9-1
Figure 1 presents images of WT and RGS9-1 -/- retinas stained to reveal the expression of RGS9-1. As reported previously, RGS9-1 antibody ("RGS9") binds strongly in the outer segment layer  in WT, but not in RGS9-1 -/- retinas. Peanut-agglutinin ("PNA") staining reveals cones at approximately the same density in normal and RGS9-1 null retinas: counts of 3 sections of 2 mice of each type yielded 2.5±0.5 (mean±s.d.) cones per 20 mm (RGS9-1 -/-) and 2.7±0.2 cones per per 20 mm (WT). Given that the sections are 15 mm thick, the cone density is estimated to be about 3 cones per (20 mm x 15 mm) = 300 mm2, or 1 cone per 100 mm2. Since the density of rods in rodent retinas is about 1 per 3 mm2, cones constitute about 3% of the photoreceptors in these retinas. The superposition of the images generated with the antibody and the peanut agglutinin ("PNA") confirms the strong expression of RGS9-1 in the cones of WT mice, as found previously in bovine cones , and absence of RGS9-1 in the knockout mice. No RGS9-1 expression could be reliably detected outside the photoreceptor layer.
Figure 2 compares WT and RGS9-1 -/- retinas for the expression of Gb5, and shows that Gb5 is very greatly reduced or absent in the outer layers of the retina of RGS9-1 -/- mice, and in particular, missing from the PNA-stained cones.
Rods of dark-adapted RGS9-1 -/- mice generate nearly normal circulating currents in situ but are very slow to recover from strong light stimuli
Figure 3 shows ERGs elicited from a WT and a RGS9-1 -/- mouse with a flash estimated to isomerize ~1% of the rhodopsin. The traces of Figure 3A,C, were obtained when the animals were dark-adapted overnight, for at least 12 h. The initial corneal-negative component of these traces is the a-wave (violet-highlighted portion of traces). The immediately following positive-going potential is a mixture of the rod- and cone-driven b-waves, and oscillatory potentials. Measured as in Figure 3A,C, the saturating a-wave amplitude of dark-adapted RGS9-1 -/- mice (248±66 mV, n=19) was indistinguishable from that of WT C57BL/6 mice (243±70 mV, n=16). In WT mice, more than 95% of the saturating a-wave amplitude results from suppression of rod circulating current [11,12]. Thus, the rods of fully dark-adapted RGS9-1 -/- mice generate circulating currents of normal magnitude, consistent with previous evidence from suction electrode recordings of isolated rods .
In contrast to its normal amplitude, the recovery of the a-wave of RGS9-1 -/- mice was greatly retarded relative to that in WT. In the experiments of Figure 3, after exposure to the initial flash, the mice were left to dark adapt for 2 min and then stimulated again with the same flash (traces of Figure 3C,D). Whereas the ERG of the WT mouse had a completely recovered a-wave (and thus, rod circulating current), that of the RGS9-1 -/- mouse exhibited only a very small a-wave (~10 mV; red highlight), and a corneal-positive b-wave of substantially reduced-amplitude. Previous analysis has shown that the ERGs recorded under conditions such as illustrated in Figure 3D originate exclusively from cone-driven neurons; we now proceed to characterize such responses in RGS9-1 -/- mice.
Both UV and M-cone-driven retinal responses are functional in RGS9-1 -/- mice
WT mice have both midwave (M)-sensitive (lmax=~510 nm) and UV-sensitive cones (lmax=~355 nm) [12,15,16], so we inquired as to whether the sensitivity of the cone-driven b-wave responses of RGS9-1 -/- animals are comparable to those of WT mice. Figure 4 presents two series of ERGs from an RGS9-1 -/- mouse, elicited by monochromatic 361 and 513 nm flashes of varied intensity under cone-isolation conditions. The amplitudes of the b-wave responses were extracted from filtered traces (smooth colored or gray curves). This was done to obviate potential artifacts that the oscillatory potentials might cause in estimation of b-wave amplitudes (compare WT and RGS9-1 -/- traces in Figure 3). The saturating amplitudes of the cone-driven b-waves for RGS9-1 -/- mice were on average about 50% larger than for WT animals, though this difference is not statistically significant (t=1.435, p<0.1, df=16). The amplitudes of the cone a-waves, which will be described in more detail below (cf. red highlighted early portions of traces in Figure 3), also were not reliably different between RGS9-1 -/- and WT (Table 1).
Intensity-response relations derived from the data of Figure 4, together with data of 6 additional RGS9-1 -/- mice, are shown in Figure 5A. From such data the ratio of the peak spectral sensitivities, SUV/SM, of the b-waves driven by the two cone types can be derived . As illustrated in Figure 5B, the sensitivity ratio of the RGS9-1 -/- mice, SUV/SM=3.0, is lower than that of WT mice, SUV/SM=5.2, but the difference between the ratios is not statistically significant (p>0.2, df=11). The apparent reason for the decreased sensitivity ratio of the RGS9-1 -/- mice is that their midwave cone sensitivity, SM, is higher than that of WT mice (Table 1), suggesting that M-cones or their secondary neurons may be primarily responsible.
Cone-driven responses of RGS9-1 -/- animals have profoundly slowed recovery from strong flashes
The paradigm used to investigate the recovery of cone-driven responses is illustrated in Figure 6. A conditioning flash sufficiently intense to temporarily suppress all cone-driven activity was followed at different interstimulus intervals (ISIs) by a probe flash of the same intensity. Recovery of the cone-driven responses of the WT mouse was found to be complete in about 1 s; in contrast, in the RGS9-1 -/- mouse, the first sign of recovery appeared only after 5 s, and complete recovery required about 100 s.
The cone-driven a-wave also recovers very slowly
Because the corneal-positive component of the ERG (the b-wave) represents field potentials generated mostly by second order neurons (reviewed in ), and because RGS9-1 is expressed in cones, a question of special interest is whether the slowed recovery was caused by prolonged activation of the cones themselves, or only due to prolonged activity in neurons downstream from the photoreceptors. To address this question we examined the behavior of the cone a-wave, which is thought to originate primarily in the suppression of cone photoreceptor circulating current [17-19], and which we have previously characterized in C57BL/6 mice [9,12]. Thus, in Figure 7 the initial portions of records from mice stimulated as in Figure 6 have been replotted on expanded time and amplitude scales to facilitate examination of the cone a-wave. Here the differences between the WT and mutant animals in the recoveries of the cone a-waves are seen to parallel the recoveries of the cone-driven b-wave in Figure 6. Caution is called for in identifying cone photoreceptors as the sole underlying generators of the cone-driven a-wave since the primate photopic a-wave has been shown to have a component originating in off-bipolar cells [20,21] and mice have been confirmed to have at least two types of cone off-bipolar cells (Dr. Peter Sterling, University of Pennsylvania, personal communication). However, since cone off-bipolars are driven through an ionotropic glutamate synaptic contact with cones, one can expect their light-stimulated currents to closely track the cone responses.
Figure 8 summarizes the results of applying the protocol of Figure 6 and Figure 7 to populations of WT and RGS9-1 -/- mice. Figure 8A plots the saturated amplitude of the cone-driven b-wave, as a function of time after the conditioning flash. Taking 0.4 s as the average time for the b-wave of WT mice to recover 50% of their dark-adapted amplitude, the cone b-wave of the RGS9-1 -/- mice is seen to recover almost 60-fold more slowly. Figure 8B plots the recovery of the cone a-wave, on a common abscissa with panel A. Again, using the recovery to 50% amplitude as a benchmark, the cone a-waves of the RGS9-1 -/- mice are seen to recover about 60-fold more slowly than those of WT mice.
Observations made with the rod a-wave in this investigation (Figure 3A) are consistent with the hypothesis that RGS9-1 and Gb5-L are essential for normal inactivation of rod transducin, confirming previous findings showing that single rods of RGS9-1 -/- mice exhibit profoundly slowed recovery of their photoresponses .
Our results support the conclusion that RGS9-1 -/- mice have a major defect in the inactivation of their cone phototransduction cascades, as follows. First, we have found RGS9-1 to be expressed in murine cones (Figure 1), as previously reported for bovine cones . Second, the greatly slowed recoveries of the a-wave component of the ERG of RGS9-1 -/- mice recorded under cone-signal isolation conditions (Figure 7, Figure 8B) point to a cone-cell locus for the recovery deficit.
Our results show that RGS9-1 -/- mice, like WT mice, have both UV- and M-cones, and secondary neurons driven by these cones (Figure 3, Figure 4), and support the conclusion that RGS9-1 is a GAP factor for cone transducin in both types of cones. This follows since all cone-driven activity of RGS9-1 -/- mice is greatly retarded in recovery from strong flashes (Figure 6, Figure 7). It is noteworthy in this context that only a single cone-specific transducin alpha-subunit, GNAT2, has been identified in the GenBank database, including human (Z18859) and murine (NM_008141) homologs.
It interesting to compare the effects of inactivating the primary shutoff mechanisms of phototransduction in cones, viz., the GRK1-dependent inactivation of cone R* and the RGS9-1-Gb5-dependent inactivation of the G*-E* complex. Cone a-wave and b-wave responses of GRK1 -/- mice, when tested under the same conditions used in this investigation, reach 50% recovery in about 19 s , which is comparable to the recovery half-time of 23 s in RGS9-1 -/- mice (Figure 7). Thus, though these inactivating proteins work at distinct stages of the cone transduction cascades, their deletions produce defects in recovery of comparable magnitude.
While the primary proteins of the transduction cascades of cones are established to be distinct isoforms from those of rods. Many of the proteins involved in activation and recovery are shared. The list of shared proteins established to function in both cell types now includes GRK1 (mice; , RGS9-1 (this paper)) and guanylyl cyclase, GC1 (GC-E) [22,23]. In addition, the guanylyl cyclase activating protein, GCAP1, has been established to be present in both rods and cones of mice . Given the established capacity of GCAP1 to activate GC1 (reviewed in [25-27]), and its likely role in autosomal dominant cone dystrophy , it seems certain that GCAP1 also functions in both cell types. Our results add RGS9-1 to this growing list of regulatory proteins of phototransduction shared by rods and cones.
Supported by NIH grants to ENP, TGW, and MIS, and by awards from the Research to Prevent Blindness Foundation to ENP and TGW. We thank Ann Milam (Department of Ophthalmology, University of Pennsylvania) and Trevor Lamb (Department of Physiology, University of Cambridge), and the reviewers, for helpful comments.
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