Regulation of rod photoreceptor function by farnesylated G-protein γ-subunits

Heterotrimeric G-protein transducin, Gt, is a key signal transducer and amplifier in retinal rod and cone photoreceptor cells. Despite similar subunit composition, close amino acid identity, and identical posttranslational farnesylation of their Gγ subunits, rods and cones rely on unique Gγ1 (Gngt1) and Gγc (Gngt2) isoforms, respectively. The only other farnesylated G-protein γ-subunit, Gγ11 (Gng11), is expressed in multiple tissues but not retina. To determine whether Gγ1 regulates uniquely rod phototransduction, we generated transgenic rods expressing Gγ1, Gγc, or Gγ11 in Gγ1-deficient mice and analyzed their properties. Immunohistochemistry and Western blotting demonstrated the robust expression of each transgenic Gγ in rod cells and restoration of Gαt1 expression, which is greatly reduced in Gγ1-deficient rods. Electroretinography showed restoration of visual function in all three transgenic Gγ1-deficient lines. Recordings from individual transgenic rods showed that photosensitivity impaired in Gγ1-deficient rods was also fully restored. In all dark-adapted transgenic lines, Gαt1 was targeted to the outer segments, reversing its diffuse localization found in Gγ1-deficient rods. Bright illumination triggered Gαt1 translocation from the rod outer to inner segments in all three transgenic strains. However, Gαt1 translocation in Gγ11 transgenic mice occurred at significantly dimmer background light. Consistent with this, transretinal ERG recordings revealed gradual response recovery in moderate background illumination in Gγ11 transgenic mice but not in Gγ1 controls. Thus, while farnesylated Gγ subunits are functionally active and largely interchangeable in supporting rod phototransduction, replacement of retina-specific Gγ isoforms by the ubiquitous Gγ11 affects the ability of rods to adapt to background light.


Introduction
The high sensitivity of rod photoreceptors is achieved by the activation of multiple copies of the heterotrimeric G-protein, Gt, by a single rhodopsin [1]. The Gtβγ (Gβ 1 γ 1 ) complex is a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Use Committee and the Washington University Animal Studies Committee. Unless otherwise specified, all mice were age-matched 2-to 3-month-old littermates of either sex; they were kept under the standard 12 h dark/light cycle and dark-adapted overnight before all experiments.
We introduced three individual mouse Gγ-subunits into Gγ 1 -deficient rods [18]. All transgenic constructs included the 4.4 kb mouse opsin promoter (generous gift from Dr. Lem, Tufts Medical Center) [19], mouse Gngt1 cDNA, as well as appropriate intron and poly(A) sequences (Fig 2). An in-frame insertion of 3xFLAG-HA epitope at the N-terminus of all Gγ was designed to help with detection and quantification of the expressed proteins. The following nucleic acid sequence was present in all individual synthetic genes used to generate the three transgenic constructs: tttaaactgcagaagttggtcgtgaggcactgggcaggtaagt atcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaag actcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctcca caggtgtccactcccagttcaattacagctcttaaggctagagtacttaatacgactcacta taggctagcctcgatcgagaattcacgcgtcttccctgacagaagatggactacaaagacca tgacggtgattataaagatcatgacatcgattacaaggatgacgatgacaagcttgcggccg cgaattcatacccatacgacgtaccagattacgct.

Western blotting and antibodies
Retinas from 2-month-old dark-adapted mice were dissected, flash-frozen in liquid nitrogen, and stored at -80˚C until protein quantification or biochemical experiments. Bio-Rad precast 12% Mini-Protean TGX were used for all SDS-gels. Protein transfer was performed using Trans-Blot SD semi-dry cell on PVDF membrane. Rabbit antibodies sc-389-Gα t1 , sc-15382rhodopsin were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse FLAG M2 F1804 were from Sigma-Aldrich. Rabbit HA TA150084 were from Origene. Rabbit PDE6A PA1-720, PDE6G PA1-723 and beta Actin PA1-16889 and secondary HRP antibodies were from Invitrogen. Rabbit antibodies against Gβ 1 and Gγ 1 were a gift from N. Gautam (Washington University, St. Louis, MO). Primary antibody dilution was 1:1,000. Secondary antibody dilution was 1:10,000. All gels/blots were developed and analyzed in compliance with the digital image and integrity policies. Prior to blocking non-specific binding by 5% BSA in TBST, the PVDF membranes were cut to size using Amersham Rainbow molecular weight markers as a guide. For proteins with significantly different molecular weights, such as Gα t1 and Gγ 1 , the membrane was cut in half horizontally into the upper and lower portions, which were stained with individual antibodies. After staining with primary and secondary antibodies, blots were developed using Amersham ECL Prime detection kit. Chemiluminescence was visualized using Li-COR C-DiGit 1 Blot Scanner that was setup to collect and save time-lapse data in the high-sensitivity mode. Quantitation was performed using Image Studio software. The pixel saturation tool was used to ensure that optical density (OD) of protein bands is not saturated, and only unsaturated bands in a linear range of protein band intensities were used for quantitation. Local background was subtracted.

Light microscopy and immunohistochemistry
For immune labeling, eyes were cryo-preserved in Tissue-Tek O.C.T. compound. Semi-thin 0.9-μm sections were cut in the dorsal-to-ventral direction through the optic nerve and immunostained as previously described [20]. Images were taken on a Leica DM 5500 D microscope using DFC360 FX camera. For the Gα t1 translocation experiment, mice were dark-adapted overnight, their eyes were dilated with one drop of 1% atropine sulfate and then exposed for 15 minutes to steady white background light of various intensities, measured by Sper Scientific Advanced Light Meter 840022, followed by euthanasia by CO 2 and eye cryo-preservation. Unsaturated pictures of cross-sections of the retina immunolabelled with anti-Gα t1 antibody were analyzed in Adobe Photoshop CS4 Extended using the analysis module. Integrated density (ID) was measured in the rod outer segment (OS), and combined area of rod inner segment (IS), rod outer nuclear layer (ONL) and outer plexiform layer (OPL) in three independent sections. ID OS +(ID IS +-OD ONL +OD OPL ) was taken as 100% followed by the calculation of the proportion of Gα t1 in OS as ID OS in percent.

In vivo electroretinography (ERG)
Animals were dark-adapted overnight and anesthetized by subcutaneous injection of ketamine (80 mg/kg) and xylazine (15 mg/kg). Pupils were dilated with 1% atropine sulfate. During testing, a heating pad controlled by a rectal temperature probe maintained body temperature at 37-38˚C. Full-field ERGs were recorded using a UTAS BigShot apparatus (LKC Technologies) and corneal cup electrodes, as described [21]. The reference electrode needle was inserted under the skin at the skull. Test flashes of white light ranging from 2.5x10 -5 cd�s m -2 to 700 cd�s m -2 were applied in darkness (scotopic conditions). Responses from several trials were averaged and the intervals between trials were adjusted so that responses did not decrease in amplitude over the series of trials for each step. The recorded responses were low-pass filtered at 500 Hz.

Single-cell suction recordings
Mice were dark-adapted overnight, sacrificed by CO 2 asphyxiation, and their retinas were removed under infrared illumination. Retinas were chopped into small pieces with a razor blade and transferred to a perfusion chamber on the stage of an inverted microscope. A single rod outer segment on the edge of a retina piece was drawn into a glass microelectrode filled with solution containing 140 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl 2 , 1.2 mM CaCl 2 , 3 mM HEPES (pH 7.4), 0.02 mM EDTA, and 10 mM glucose. The perfusion solution contained 112.5 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl 2 , 1.2 mM CaCl 2 , 10 mM HEPES (pH 7.4), 20 mM NaHCO 3 , 3 mM Na succinate, 0.5 mM Na glutamate, 0.02 mM EDTA, and 10 mM glucose. The solution was bubbled with 95% O 2 / 5% CO 2 mixture and its temperature was maintained at 37˚C with an in-line ceramic heater.
Rods were stimulated with 20-ms test flashes of calibrated 500 nm light. The light intensity was controlled with neutral density filters in 0.5 log unit steps. Photoresponses were amplified, low-pass filtered (30 Hz,, and digitized (1 kHz). Data were analyzed using Clampfit 10.6 and Origin 8.5 software. Intensity-response relationships were fitted with Naka-Rushton hyperbolic function: where R is the transient-peak amplitude of the rod response, R max is the maximal response amplitude, I is the flash intensity, n is the Hill coefficient (exponent), and I 1/2 is the half-saturating light intensity. Normalized rod flash sensitivity (S f ) was calculated from the linear part of the intensity-response curve, as follows: where R is the amplitude of dim flash response, R max is the maximal response amplitude for that cell, and I is the flash strength used to elicit the dim flash response. The amplification of the rod phototransduction cascade was evaluated from test flash intensities that produced identical rising phases of dim flash responses. This approach was preferred to calculation of the amplification constant by the method of Lamb and Pugh [22], due to the relatively long duration of test flashes and the effect of low-pass filtering on the response front. Integration time (T integr. ) was calculated as the integral of the dim flash response with the transient peak amplitude normalized to unity. The time constant of the dim flash response recovery (τ rec ) was derived from single-exponential fit to the falling phase of the response. The dominant recovery time constant (τ D ) was determined from supersaturating flashes [23], using a 10% criterion for recovery of the photocurrent from saturation.

Transretinal ERG recordings
Mice were dark-adapted overnight and sacrificed by CO 2 asphyxiation. The whole retina was removed from each mouse eyecup under infrared illumination and stored in oxygenated aqueous L15 (13.6 mg/ml, pH 7.4) solution (Sigma-Aldrich) containing 0.1% BSA, at RT. The retina was mounted on filter paper with the photoreceptor side up and placed in a perfusion chamber [24] between two electrodes connected to a differential amplifier. The tissue was perfused with bicarbonate-buffered Locke's solution supplemented with 2 mM L-glutamate and 10 μM DL-2-amino-4-phosphonobutyric acid to block postsynaptic components of the photoresponse [25], and with 20 μM BaCl 2 to suppress the slow glial PIII component [26]. The perfusion solution was continuously bubbled with a 95% O 2 / 5% CO 2 mixture and heated to 36-37˚C.
The photoreceptors in the retina were stimulated with 20-ms test flashes of calibrated 505 nm LED light. The light intensity was controlled by a computer in 0.5 log unit steps. The prolonged (> 1 h) background illumination was achieved with the same 505 nm LED activating 830 rhodopsin molecules (R � ) per rod per second initially. Photoresponses were amplified by a differential amplifier (DP-311, Warner Instruments), low-pass filtered at 30 Hz (8-pole Bessel), and digitized at 1 kHz. Data were analyzed with Clampfit 10.6 and Origin 8.5 software.

Statistical analysis
For all experiments, data were expressed as mean ± SEM and analyzed with the independent two-tailed Student's t-test (using an accepted significance level of p < 0.05).

Generation of the three transgenic Gγ lines
The transgenic mice were generated using the construct shown in Fig 2. We used the mouse opsin promoter to target the expression of each of the three transgenic Gγ subunits selectively in rod photoreceptors. We also included a 3xFLAG and an HA tag to facilitate detection of the transgenic protein in the retina. Upon the successful generation of the three Gγ 1 , Gγ c , and Gγ 11 transgenic strains, we crossed them with the rod Gγ 1 -deficient (Gngt1 -/-) line to effectively substitute the rod Gγ 1 with each of the transgenic Gγ subunits. As we have shown previously, deletion of rod Gγ 1 in mice results in dramatic suppression of rod sensitivity and reduction in the expression of the other two rod transducin subunits, Gα t1 and Gβ 1 [2], see also [3]. Thus, generating Gγ 1 + Gngt1 -/-, Gγ c + Gngt1 -/-, and Gγ 11 + Gngt1 -/mice allowed us to investigate how the substitution of the endogenous rod Gγ 1 subunit with transgenic Gγ 1 (as a control), or with Gγ c or Gγ 11 will affect the Gt expression profile and functional properties of mouse rods.
We began our analysis by investigating the expression localization of the Gγ 1 , Gγ c , and Gγ 11 γ-subunits in their respective transgenic mouse retinas. To prevent light-driven translocation and ensure that all Gt subunits were properly localized in the outer segments of rods, these experiments were performed after dark-adapting the animals overnight. Using an anti-FLAG antibody staining of retinal sections, we found, as expected, that no transgenic protein was found in wild type or Gngt1 -/retinas ( Fig 3A and 3B). Transgenic Gγ 1 , Gγ c , and Gγ 11 subunits were all, indeed, localized in the outer segments of rods (Fig 3C-3E). Thus, in addition to the transgenically reintroduced Gγ 1 , both cone Gγ c and the non-photoreceptor Gγ 11 were targeted properly to the rod outer segments following dark adaptation.
The level of transducin in rod outer segments is directly proportional to the amplification of rod phototransduction [27], making its proper translocation crucial for the function of rods. Our finding that all three transgenic Gγ subunits localized properly to the rod outer segments was critical for enabling us to perform the subsequent physiological analysis of the three transgenic mouse lines and to compare directly their functional properties. Notably, our immunohistochemical analysis also showed that all three transgenic lines retained normal retina morphology and uniform expression of the transgenic proteins in the Gγ 1 -deficient rods.

Restoration of transducin complement in all Gγ-expressing lines
Quantitative Western blot analysis was performed in the linear portion of the dose escalation plots of the total retina protein vs. optical densities of the protein bands to assure the Western

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Farnesylated G-protein gamma-subunits in transgenic rods signal is not saturated, typically in the 5-20 μg range. It showed that expression levels of general cellular protein actin and rhodopsin in the retina were comparable in Gγ 1 + Gngt1 -/-, Gγ c + Gngt1 -/-, and Gγ 11 + Gngt1 -/mice ( Fig 4A and 4B), a finding consistent with the normal morphology and lack of degeneration in these retinas (Fig 3). Direct protein expression comparison in Fig 4C used 10 μg of retina protein in each sample. Gγ 1 , Gγ c , and Gγ 11 transgenic proteins were easily identified by both anti-FLAG and anti-HA staining (Fig 4C). Expression levels of the three γ-subunits also appeared similar by this test. Gγ 1 -specific antibodies stained transgenic Gγ 1 stronger, compared to the native Gγ 1 in WT samples (Fig 4C, bottom), which may be explained either by higher level of transgenic protein whose expression is driven by the strong rhodopsin promoter compared to the Gngt1 promoter in wild type retinas, or possibly by better accessibility of the N-terminal epitope in the transgenic protein. Western blots also showed that expression of each of the transgenic Gγ subunits restores the amounts of Gα t1 to wild type levels ( Fig 4C). Restoration of Gα t1 expression in all transgenic lines was also corroborated by the robust staining and proper Gα t1 localization to the rod outer segments in dark adapted retinas, discussed separately in Fig 8. The expression levels of Gβ 1 were also recovered ( Fig 4C). As expected, all three transgenic retinas expressed equal amounts of the effector protein PDE6, as judged by the similar intensities of protein bands for PDE6α and PDE6γ

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Farnesylated G-protein gamma-subunits in transgenic rods ( Fig 4C). Thus, transgenic retinas appeared to express the full and equal sets of rhodopsin, transducin, and PDE.

Restoration of scotopic visual function in all Gγ-expressing lines
To determine how the expression of each of the three Gγ-subunits affects the functional properties of rods, we first performed electroretinography (ERG) analysis of control wild type and Gngt1 -/mice and the transgenic Gγ 1 + Gngt1 -/-, Gγ c + Gngt1 -/-, and Gγ 11 + Gngt1 -/mice in vivo (Fig 5A-5E). As we have previously shown [2], deletion of the rod Gγ 1 -subunit results in substantial desensitization and reduction in the maximal ERG a-wave response (Fig 5F, open

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circles) all restored robust scotopic function essentially to the wild type level (Fig 5F, open black circles; see also [28] for the reference to wild type data). Thus, not only did the transgenic expression of Gγ 1 rescue scotopic vision in the Gγ 1 -deficient mice, but the same effect could be achieved by expressing the cone Gγ c or the non-photoreceptor Gγ 11 .

Restoration of rod photosensitivity and response kinetics in all Gγexpressing lines
Next, we analyzed by suction electrode recordings whether the transgenic expression of the three different Gγ-subunits in individual Gngt1 -/mouse rods would restore their photosensitivity and response kinetics. In agreement with the similar length of their outer segments at the age of 4-5 weeks (Fig 2) and normal ERG responses in vivo (Fig 5), Gγ 1 + Gngt1 -/-, Gγ c + Gngt1 -/-, and Gγ 11 + Gngt1 -/rods produced saturated responses of similar amplitudes, not different from these in wild type and Gngt1 -/cells (Fig 6A-6F and Table 1). Remarkably, compared to the dramatically desensitized (~70-fold) Gγ 1 -deficient rods, the light sensitivity of all transgenic photoreceptors was restored to wild type levels ( Fig 6F). It should be noted, however, that the average sensitivity of Gγ 11 + Gngt1 -/rods was slightly (~20%) higher than that in the other two Gγ-expressing lines (Table 1).
We then evaluated the kinetics of activation of the rod phototransduction cascade in all three mutant mouse strains by directly comparing the light intensities required to produce identical initial phases of response activation (Fig 7A). In accordance with their restored sensitivity, the phototransduction amplification in Gγ 1 + Gngt1 -/rods was increased by~34-fold compared to that in cells lacking Gγ 1 and reached wild type level, as evident from the analysis of rising phases of their dim flash responses during the first 40 ms after the test flash. The cascade activation was only slightly (~10%) lower in Gγ c + Gngt1 -/rods and higher (by~10%) in Gγ 11 + Gngt1 -/cells than in the Gγ 1 -expressing transgenic rods, thus showing a comparable degree of restoration in all three transgenic lines.
One characteristic feature of Gngt1 -/rods is the significantly faster inactivation of their signaling cascade, an effect contributing to their reduced photosensitivity [2]. In contrast, normal inactivation rate of dim flash responses was achieved in the rods of all transgenic lines expressing a Gγ-subunit, as judged from their normal time-to-peak, integration time, and singleexponential dim flash response recovery time constant (τ rec ) ( Fig 7B and Table 1). Coincidentally, the response recovery following supersaturating flashes was also slower in all transgenic lines than in Gγ 1 -deficient controls, as evident from comparing the kinetics of their maximal rod responses (Fig 7C) and the corresponding dominant recovery time constants (τ D ) ( Fig 7D  and Table 1). All these parameters were also comparable to those typically observed in wild type mouse rods (Table 1 and [2]). It should be mentioned that the rods expressing Gγ 11 had the slowest τ D among all transgenic cells (Table 1) although the molecular mechanisms behind their slight response deceleration remain unclear. Taken together, these results indicate that the transgenic expression of various G-protein γ-subunits with distinct amino acid sequences rescues equally well the expression level of rod transducin α-subunit in Gγ 1 -deficient mouse rods and effectively restores their signaling, although with slightly different photoresponse kinetics.

Light-driven translocation of Gtα 1 in Gγ-expressing rods
Finally, we investigated how the expression of each of the three transgenic Gγ subunits in rods affects the light-driven translocation of Gα t1 from the outer segment to the inner segment of these photoreceptors. We examined the distribution of Gα t1 across the rods in 5 different background light conditions: darkness and at 1, 10, 100, and 1000 lux of steady background

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Farnesylated G-protein gamma-subunits in transgenic rods illumination. To allow translocation to occur, dark-adapted animals were exposed to the background light for 15 minutes, and then were rapidly euthanized and their eyes were dissected, cryo-preserved, sectioned, and stained with the Gα t1 antibody for immunohistochemical Gγ c + Gngt1 -/-(n = 30), Gγ 11 + Gngt1 -/-(n = 24), and wild type (n = 8) mouse rods. Data were fitted with hyperbolic Naka-Rushton functions that yielded half-saturating light intensities (I 1/2 ) indicated in Table 1. Error bars are smaller than the symbol size for most data points.

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Farnesylated G-protein gamma-subunits in transgenic rods analysis of its distribution. Consistent with the localization of the transgenic Gγ 1 , Gγ c , and Gγ 11 subunits to the outer segments of rods in dark-adapted retinas (Fig 3), we found that Gα t1 was also properly localized in the rod outer segments in darkness (0 lux; Fig 8A-8C, left panels, and 8D). In Gγ 1 + Gngt1 -/and Gγ c + Gngt1 -/mice, approximately 90% of Gtα t1 remained in the outer segments in dim background illumination of 1 and 10 lux, and eventually translocated to the inner segments when the retinas were illuminated with 100 and 1000 lux of light (Fig 8A and 8B, right two panels). This is qualitatively consistent with previous work showing that in wild type mouse rods the threshold for transducin translocation is near 4.6x10 3 R � rod -1 s -1 [29], and indistinguishable from the Gtα t1 translocation in wild type and Gngt1 +/retinas under identical conditions. The Gngt1 +/control contains one Gngt1-wild type copy and one Gngt1copy and could be used as a closer genetic match for Gγ 1 + Gngt1 -/containing one copy of the Gngt1 transgene and two Gngt1copies. In contrast, translocation of Gα t1 in Gγ 11 + Gngt1 -/retinas was triggered with illumination as low as 1 lux (Fig 8C and 8D, blue circles). At 1 lux, only 10% of Gα t1 remained in the outer segments of the Gγ 11 + Gngt1 -/retinas compared to 90% for the other two Gγ transgenes in respective lines (Fig 8D). The highly robust Gα t1 staining in the outer nuclear layer that is evident at 100 and 1000 lux in the Gγ 11 + Gngt1 -/retinas is typically observed in wild type and Gngt1 +/controls only at background illumination levels above 1000 lux. Thus, surprisingly, despite the essentially identical functional properties of dark-adapted rods expressing the three transgenic Gγ subunits, translocation of transducin during continuous light exposure was initiated at substantially lower light intensity in transgenic Gγ 11 rods compared to transgenic Gγ 1 or Gγ c cells.
It was recently shown that the gradual translocation of transducin from the outer to the inner segments of rods under continuous illumination results in partial recovery of the rod response after its initial suppression by the background light [30]. Thus, we sought to determine whether the lower threshold for Gα t1 translocation found in Gγ 11 + Gngt1 -/retinas affects the amplitude of the rod response over the course of 1-h exposure to background light. We used transretinal (ex vivo ERG) recordings to obtain and monitor the rod-driven responses. We exposed control Gngt1 +/and transgenic Gγ 11 + Gngt1 -/retinas to a moderate sub-saturating background light activating~830 visual pigment molecules (R � ) per rod per second at onset. This light would be expected to trigger transducin translocation in Gγ 11 transgenic retinas but not in control retinas (Fig 8, see also [29]). As expected, in control retinas, the onset of R max , maximal dark current measured from saturated responses; time-to-peak (T peak ), integration time (T integr. ), and normalized flash sensitivity (S f ) refer to responses whose amplitudes were *0.2�R max and fell within the linear range; I 1/2 , half-saturating light intensity; τ rec , time constant of single-exponential decay of the dim flash response recovery phase; τ D , dominant time constant of recovery after supersaturating flashes determined from the linear fit to time in saturation vs. intensity semilog (Pepperberg) plots [23].

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Farnesylated G-protein gamma-subunits in transgenic rods the background light caused a rapid partial suppression of the rod maximal response (Fig 9,  black symbols), which then persisted largely unchanged for the 60-min duration of the experiment, only slightly affected by a gradual rundown. The onset of an identical background light in Gγ 11 + Gngt1 -/retinas produced comparable initial suppression of the rod maximal response.
However, in stark contrast to the control case, the rod response then gradually recovered over the course of the 60 min of the experiment (Fig 9, blue symbols). As recently argued, this gradual increase reflects the translocation of Gα t1 away from the rod outer segments, which would effectively reduce the activation of the rod phototransduction by the steady background light, allowing the rods to recover partially their dark current [30]. Thus, the gradual recovery of rod responses in transgenic Gγ 11 + Gngt1 -/retinas but not in control retinas in moderate background light is consistent with our observation that in these conditions transducin translocation takes place only in the transgenic Gγ 11 + Gngt1 -/rods but not in controls (Fig 8).

Discussion
Heterotrimeric G-proteins are the main transducers and amplifiers of extracellular signals from GPCRs to the intracellular effectors. It is now firmly established that specificity of the GPCR signaling and fine-tuning of the resulting physiological responses are regulated by the diversity of the Gα subunits, comprised of sixteen family members subdivided into four subfamilies (G s , G i/o , G q/11 , and G 12/13 ), as well as by multiple combinations of five Gβ (Gβ 1-5 ) and twelve Gγ (Gγ 1-13 ) subunits. In many cell types containing various G-protein combinations, their interplay contributes to the rich gamut of cellular responses with defined spatiotemporal characteristics.
Retinal rod and cone photoreceptors provide a fascinating example of highly specialized sensory neurons that, while employing similar signaling architecture, differ drastically in their light sensitivity, photoresponse kinetics, and light adaptation properties. Being on the other side of the spectrum from a typical cell that contains multiple G-protein types, rods and cones rely on conserved cell-specific G-protein heterotrimers: Gα t1 /Gβ 1 γ 1 and Gα t2 /Gβ 3 γ c , respectively [31]. While trace expression levels of Gγ 2 and Gγ 3 subunits were detected in rods, their physiological contribution in phototransduction is negligible [32]. This property makes rods a unique model system to study the physiological roles of G-protein subunits in visual transduction by substituting individual rod-specific G-protein subunits with their cone-specific or ubiquitous isoforms. This experimental design was successful to show that when Gα t1 was replaced by Gα t2 in rods, while retaining native rod Gβ 1 γ 1 complex, the phototransduction was largely unaffected [5][6][7].
To determine the physiological role of Gβγ in photoreceptor function, we previously genetically removed the gene Gngt1 encoding rod Gγ 1 subunit and demonstrated that the high light sensitivity of rods and their robust signal amplification are severely compromised in mice [2]. The Gngt1 -/model provided an excellent starting point to pose the next question of the possible physiological difference between various Gγ isoforms. Specifically, what is the reason for the selective use of Gγ 1 and Gγ c in rods and cones, respectively, and the exclusion of otherwise ubiquitously expressed Gγ 11 from both photoreceptor types? This question is especially intriguing considering the fact that these three Gγ proteins belong to the same Class I Gγ subunits that are post-translationally modified by the shorter isoprenoid lipid farnesyl, as opposed to class II-IV Gγ subunits that are geranylgeranylated [33]. Farnesylation is required for proper targeting of G-proteins to the outer segment and full biological activity [34,35]. Thus, replacing native rod Gγ 1 with cone Gγ c or Gγ 11 subunit ensures highly controlled experimental conditions not affected by the Gγ class or isoprenylation differences.
Here, we generated three individual transgenic mouse lines expressing Gγ c , Gγ 11 , and control Gγ 1 on the Gngt1 -/background (Fig 2). Immunohistochemical staining of retina crosssections for the FLAG epitope that was included in all transgenic constructs showed similarly healthy retina morphology, uniform expression of these Gγ proteins and their proper targeting to the rod outer segments (Fig 3). The levels of expression of other major phototransduction proteins, such as rhodopsin, transducin subunits, and PDE were identical between the experimental and control retinas (Fig 4). Transgenic re-introduction of Gγ 1 , Gγ c , or Gγ 11 also completely restored the levels of endogenous Gα t1 (Fig 4) that is known to be severely reduced by the deletion of native Gγ 1 [2,3]. This result is of particular importance because signal amplification in mammalian rods is directly proportional to the level of expression of their Gα t1 subunit [27]. Thus, morphological and protein expression data argue that rods from the Gγ 1 , Gγ c , and Gγ 11 transgenic lines are indistinguishable in their structure and protein complement.
Because Gβγ complexes function natively as inseparable heterodimers, the deletion of Gγ 1 in rods is expected to lead to accumulation of misfolded Gβ 1 protein. Slow progressive retinal degeneration in the Gγ 1 deficient mice was proposed to be the result of proteostatic stress, or inability of the rod cell ubiquitin-proteasome system to degrade un-complexed Gβ 1 protein effectively [36][37][38][39]. Expression of Gγ 1 , Gγ c , and Gγ 11 in the Gγ 1 deficient mice appears to rescue the retina degeneration phenotype independent of the type of the Gγ subunit, which argues for the productive complex formation of Gβ 1 γ 1 , Gβ 1 γ c , and Gβ 1 γ 11 dimers and confirms previous biochemical results [40]. In addition, equal levels of the Gα t1 expression in transgenic retinas (Fig 4) and effective delivery of Gα t1 to the rod outer segments under dark adapted conditions (Fig 8) are consistent with normal heterotrimer formation and its proper subcellular localization.
There is a growing body of evidence that Gβγ-complexes contribute to the complexity and diversity of GPCR-mediated signaling that is shaped by specificity and response kinetics of GPCR/G-protein interactions at the plasma membrane, via direct interactions with effector molecules, as well as by acting at distant sites such as intracellular organelles [40,41]. Thus, we examined whether Class I Gγ 1 , Gγ c , and Gγ 11 modified by posttranslational farnesylation (Fig  1) would restore scotopic visual function, and to what extent they would determine rod photosensitivity and response kinetics. This question is especially intriguing while comparing and contrasting rod Gγ 1 and cone Gγ c , as retinal rods respond to light at significantly lower light levels compared to cones, and rod response kinetics are markedly slower [42]. The results from our in vivo ERG experiments and single-cell suction electrode recordings conclusively demonstrate that despite minor variations, all three Class I Gγ subunits can support essentially normal scotopic rod photoresponses (Figs 5-7). Thus, the differences in Gγ composition between rods and cones cannot explain their unique activation properties in dark-adapted conditions. This also implies that Gγ involvement in the activation properties of photoreceptors per se has unlikely contributed to the evolutionary selection of Gγ 1 for rods, Gγ c for cones, and Gγ 11 for other tissues. The physiological features determining selective expression of Gγ 1 and Gγ c in rods and cones is still to be determined. Our results mirror a previous observation obtained by replacing rod Gα t1 by cone Gα t2 that these two Gα t isoforms are functionally interchangeable [5]. Knowing that neither Gα t2 nor Gγ c makes the rod cascade activation cone-like, it remains quite possible that unique properties of cone phototransduction are determined by the Gγ c counterpart Gβ 3 as part of the unique cone Gβ 3 γ c complex, as deletion of Gβ 3 alone in cones doesn't affect cone response kinetics [43]. Alternatively, differences in upstream and downstream phototransduction components [44][45][46], as well as structural differences between rods and cones could account for their unique functional characteristics.
In stark contrast to the functional interchangeability of Gγ 1 , Gγ c , and Gγ 11 in dark-adapted rod phototransduction, we observed a significant effect by the Gγ composition on the cell responsiveness in steady background light. Upon increasing the intensity of background illumination rod responses saturate quickly, the process accompanied by massive light-driven translocation of Gα t1 from the rod outer to the rod inner segment [27]. While Gα t1 translocation was similar in Gγ 1 and Gγ c transgenic retinas, substitution of Gγ 1 with Gγ 11 shifted the light threshold that triggers translocation to lower background light intensity by 2-3 orders of magnitude (Fig 8). We observed that transducin in Gγ 11 transgenic rods began to translocate at a light intensity of just 1 Lux, while Gγ 1 and Gγ c transgenic rods were still deeply darkadapted. This remarkable effect had profound implications on rod function, as only Gγ 11 transgenic rods recovered their response amplitudes under a moderate steady background light, as observed in our transretinal ERG recordings (Fig 9).
While Gγ 11 is normally excluded from rods and cones [15], and thus transducin heterotrimer Gα t1 Gβ 1 γ 11 is likely not physiologically relevant, our results clearly demonstrate that in principle, the type of Gγ isoform can have significant implications for light adaptation and the kinetics of photoreceptors' escape from physiological saturation. Because Gγ 1 , Gγ c , and Gγ 11 belong to the same class of farnesylated Gγ subunits, the observed effect must be attributed to the unique amino acid sequence of Gγ 11 (Fig 1). Interestingly, a previous study utilizing the knock-in of the geranylgeranylated mutant of Gγ 1 demonstrated normal photoresponses but impaired photoresponse recovery caused by the stronger interaction of the mutant protein with lipid membranes and compromised light-driven translocation of Gt [47], a predictably opposite effect to what we observed with Gγ 11 . Similarly, a recent study with mutant Gα t1 that associates more strongly with Gβ 1 γ 1 and as a result does not translocate efficiently in comparable background light, showed a suppressed recovery of the rod dark current under those conditions [30]. In the context of these findings, our results suggest that Gα t1 associates more weekly with Gβ 1 γ 11 than with the endogenous Gβ 1 γ 1 , causing easier dissociation and translocation upon light exposure. This conclusion is also supported by the comprehensive biochemical analysis of the heterotrimeric G-protein complex formation that demonstrated significantly weaker association of Gβ 1 γ 11 compared to Gβ 1 γ 1 with Gα i1 , a close relative of Gα t1 [48]. Taken together, it appears that the Gγ-subunit amino acid sequence and the prenylation identity contribute to the unique physiological properties of rod photoreceptors under continuous illumination.

Conclusion
By replacing the native Gγ 1 subunit in mouse rod photoreceptors with cone-specific Gγ c or ubiquitous Gγ 11 isoforms , we examined the contribution of Gγ to the unique physiological properties of rods. Our results unequivocally show that while Class I Gγ subunits are functionally interchangeable in rod phototransduction, they control the light threshold for transducin translocation and the physiological light adaptation properties of rods.