Transducin translocation contributes to rod survivaland enhances synaptic transmission from rodsto rod bipolar cellsAnurima Majumdera,1, Johan Pahlbergb,1, Kimberly K. Boyda, Vasily Kerova,2, Saravanan Kolandaiveluc,Visvanathan Ramamurthyc, Alapakkam P. Sampathb,3, and Nikolai O. Artemyeva,d,3

Departments of aMolecular Physiology and Biophysics and dOphthalmology and Visual Sciences, University of Iowa, Iowa City, IA 52242; bZilkha NeurogeneticInstitute, Department of Physiology and Biophysics, University of Southern California, Los Angeles, CA 90089; and cDepartment of Ophthalmology andBiochemistry, West Virginia University Eye Institute, Morgantown, WV 26506

Edited by King-Wai Yau, Johns Hopkins School of Medicine, Baltimore, MD, and approved June 17, 2013 (received for review December 27, 2012)

In rod photoreceptors, several phototransduction components dis-play light-dependent translocation between cellular compartments.Notably, the G protein transducin translocates from rod outersegments to inner segments/spherules in bright light, but thefunctional consequences of translocation remain unclear. We gen-erated transgenic mice where light-induced transducin translocationis impaired. These mice exhibited slow photoreceptor degeneration,which was prevented if they were dark-reared. Physiological record-ings showed that control and transgenic rods and rod bipolar cellsdisplayed similar sensitivity in darkness. After bright light exposure,control rods were more strongly desensitized than transgenic rods.However, in rod bipolar cells, this effect was reversed; transgenicrod bipolar cells were more strongly desensitized than control. Thissensitivity reversal indicates that transducin translocation in rodsenhances signaling to rod bipolar cells. The enhancement could notbe explained by modulation of inner segment conductances or thevoltage sensitivity of the synaptic Ca2+ current, suggesting interac-tions of transducin with the synaptic machinery.

retina | adaptation | presynaptic modulation | SNARE complex |palmitoylation

Exposure of rod photoreceptors to bright light induces thetranslocation of key signaling molecules, including the G

protein transducin (1–4), arrestin (2, 3), and recoverin (5), be-tween their outer segment (OS), and inner segment (IS)/synapticterminal (spherule). Transducin’s translocation has been suggestedto contribute to light adaptation, because signal amplification inthe phototransduction cascade seems to be reduced aftertranslocation (4). However, subsequent studies revealed thattransducin translocation is only triggered by light intensities thatsaturate rods (6). Accordingly, transducin’s movement fromOS to IS might contribute to light adaptation within a very limitedrange of illumination corresponding to the transition from rod- tocone-mediated vision. However, when light intensity decreasesand vision begins to shift from cone- to rod-mediated, the reducedOS transducin concentration may allow rods to be responsiveduring the lengthy process of dark adaptation (7).Light-induced transducin translocation might also serve a

neuroprotective role (8). Indeed, during the day, when rods arelargely unresponsive, transducin’s exit from OS cuts excessiveactivation of phototransduction, perhaps reducing the efficacyof proapoptotic mechanisms that contribute to cell death (8).Consistent with this idea is the finding that slow retinal de-generation in Shaker1 mice coincides with an increased lightthreshold for translocation of rod transducin (9). However, adirect link between transducin translocation and retinal de-generation has not been established.Surprisingly, the possibility that the light-induced redistribution of

transducin may also play a role in modulation of synaptic trans-mission from rods to rod bipolar cells has received little attention.Clearly, a fraction of transducin reaches rod spherules in the outerplexiform layer in bright light and returns to theOSonly after several

hours of dark adaptation (2, 10). Furthermore, mice lacking phos-ducin, a key Gβγ binding partner, display reduced ON bipolar cellsensitivity (11). Thus, a role for G-protein activity in tuning synaptictransmission between rods and bipolar cells remains plausible.To study the functional significance of light-induced trans-

ducin translocation, we generated a transgenic mouse modelwhere this process is impaired. Activation of heterotrimerictransducin (Gαt1β1γ1) by photoexcited rhodopsin (R*) normallyfacilitates the dissociation of Gαt1GTP and Gβ1γ1 subunits fromone another and from the disk membrane, allowing both todiffuse to the IS and spherule of rods. Based on the well-studieddiffusion mechanism of this process (4, 7, 10, 12, 13), we gen-erated a mouse model where an artificial S-palmitoylation sitewas introduced into Gαt1, promoting a higher affinity for mem-branes. Indeed, previous studies of this mutant Gαt1A3C inXenopus laevis rods reveal that it remains substantially in the OSafter bright light exposure (14). Here, we present analysis of theGαt1A3C mouse model, which supports the role of transducintranslocation in rod survival and reveals a unique role in en-hancing signal transmission from rods to rod bipolar cells.

ResultsAdditional Palmitoylation Impairs Light-Dependent TransducinTranslocation in Gαt1A3C Rods. To study the role of rod trans-ducin (Gαt1) translocation in the function of rod photoreceptors,our strategy was to generate a mouse model where translocation isimpaired. Specifically, we introduced an additional S-palmitoyla-tion site into Gαt1 that is expected to anchor Gαt1 more tightly tomembranes and hinder diffusion to the IS after light exposure (14).The A3C substitution was introduced into a transgene for ex-pression of the Glu-Glu epitope-tagged Gαt1, which is controlledby the rod opsin promoter (Fig. 1A). The Glu-Glu tagging of Gαt1does not alter its biochemical activity or light-evoked responsesmeasured from mouse rods (15, 16). Transgenic mice were sub-sequently bred into a Gαt1−/− background (17) to study the phe-notype of A3C mice, which express only the mutant Gαt1A3C.Expression of Gαt1A3C in A3C mice was similar to the Gαt1

level in control Gαt1+/− mice, which express transducin at ∼80%of the level in WT mice (17) (Fig. 1B and Fig. S1). Given the roleof phosducin in light-dependent translocation of transducin (18)and regulation of the rod-to-bipolar cell signaling (11), we examined

Author contributions: A.M., J.P., V.R., A.P.S., and N.O.A. designed research; A.M., J.P., K.K.B.,V.K., S.K., A.P.S., and N.O.A. performed research; A.M., J.P., V.R., A.P.S., and N.O.A. analyzeddata; and A.M., J.P., V.R., A.P.S., and N.O.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1A.M. and J.P. contributed equally to this work.2Present address: Department of Biochemistry, University of Iowa, Iowa City, IA 52242.3To whom correspondence may be addressed. E-mail: asampath@usc.edu or nikolai-artemyev@uiowa.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1222666110/-/DCSupplemental.

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phosducin expression and localization in the A3C retina. Neitherthe expression levels nor the light-independent distribution ofphosducin in mouse rods was affected by the A3C mutation inGαt1 (Fig. S2). The levels of other key phototransduction pro-teins in A3C and control mice were also comparable (Fig. 1B). Inaddition, the retinal morphology of A3C mice seemed normal atthe age of 2–3 mo (Fig. 1C). Thus, A3C rods appear to be in-distinguishable from control rods in every respect, except for theexpression of the mutant Gαt1A3C.The S-palmitoylation status of Gαt1A3C in the A3C retina was

assessed quantitatively by a resin-assisted capture (RAC) tech-nique (19). Native retinal Gαo, a protein known to be palmitoy-lated, served as positive control. Palmitoylation was dependenton hydroxylamine cleavage. This analysis showed that ∼40% ofGαt1A3C was palmitoylated, which was similar to the palmitoyla-tion level of Gαo (Fig. 1D). No palmitoylation of Gαt1 was detectedin control mice (Fig. 1D). To examine the effect of Gαt1A3Cpalmitoylation on its interaction with membranes, we performedextraction of bleached A3C and control retinal membranes withisotonic buffer containing GTPγS. The membrane-bound proteinswere subsequently extracted with 1% Triton X-100 followed byextraction with 1% SDS. As expected, the dominant fraction ofGαt1 (>90%) from control retinas was found in the GTPγS extract(Fig. 1E). In contrast, ∼50% of Gαt1A3C remained in the mem-brane fraction. A small portion of the membrane-bound Gαt1A3Cassociates with the Triton X-100–resistant fraction (Fig. 1E).

Localization of transducin in dark-adapted A3C and controlmice was essentially indistinguishable. Both Gαt1A3C and Gβ1γ1were correctly targeted to the OS (Fig. 2 A and B). Translocationof transducin was assessed in mice exposed to 500 lx light for 45min after their pupils were dilated (Materials and Methods and SIText). In control mice, the bulk of transducin moved in light tothe IS and other rod compartments (Fig. 2 A and B and Figs. S3and S4). However, transducin translocation in A3C mice wasclearly impaired. The main fractions of Gαt1A3C (∼47%) andGβ1γ1 (∼44%) were retained in the OS (Fig. 2 A and B and Figs.S3 and S4). However, light-induced IS→OS redistribution ofarrestin was not altered in A3C mice (Fig. S5).

Dark Rearing Protects A3C Mice from Retinal Degeneration. Al-though the retinal morphology of A3C mice maintained on a12-h light/dark cycle was normal up to 3 mo of age, by the ageof 4 mo, they developed retinal degeneration (Fig. 2 C and D).The thickness of the outer nuclear layer (ONL) of A3C mice wasreduced from ∼45 μm at 2 mo to ∼31 μm at 4 mo of age (Fig. 2C).The subsequent progression of retinal degeneration in A3C mice

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Fig. 1. Characterization of A3C mouse retina. (A) Transgenic construct forgeneration of A3C mice. (B) Immunoblot analysis of key phototransductionproteins. Samples contained 5, 10, and 20 μg retinal homogenate from 2-mo-oldA3C (Gαt1A3C/Gαt−/−) or control Gαt1+/− mice. The antibodies are described inMaterials and Methods and SI Text. (C) Retinal morphology of 2-mo-old controland A3C mice. INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outerplexiform layer. (Scale bar: 50 μm.) (D) Palmitoylation of Gαt1A3C using resin-assisted capture assay. Immunoblot (Upper Left) with indicated antibodies showsthe elution of palmitoylated Gαo after hydroxylamine (+HA) cleavage. In con-trast, native transducin (Gαt1) is not observed in these blots. In retinal extractsfrom A3C animals (Upper Right), both Gαo and Gαt1A3C are eluted only inpresence of HA. Lower depicts the quantitative measure of observed proteinpalmitoylation based on elution from the resin. E, elution; T, total; UB, unbound.(E) Membrane association of Gαt1 in control and A3C mouse retinas. SolubleGTPγS-extracted, membrane Triton X-100–extracted, and Triton X-100 insolubleSDS-extracted fractions were obtained from mouse retinas as described inMaterials and Methods and SI Text. The fractions were analyzed by Westernblotting with anti-Gαt1 antibodies K-20 (Santa Cruz Biotechnology); 1×, 4×, and25× correspond to 4%, 16%, and 100% of one-retina fractions, respectively.

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Fig. 2. Impaired light-dependent translocation of transducin and retinaldegeneration in A3C mice. Localization of (A) Gαt1 and (B) Gβ1γ1 in dark-adapted (D) and light-exposed (L; 45 min, 500 lx) control Gαt1+/− and A3Cmice. Retina cryosections were stained with anti-Gαt1 or Gβ1 antibodies andcounterstained with Quinolinium, 4-[3-(3-methyl-2(3H)-benzothiazolyli-dene)-1-propenyl]-1-[3-(trimethylammonio)propyl]-, diiodide (TO-PRO3)nuclear stain (blue in D panels). (Scale bar: 20 μm.) (C) Progression of retinaldegeneration in A3C mice. The ONL thickness (mean ± SEM) was determined infour animals of each age group. *P = 0.011; **P = 0.0017; ***P = 0.0002; ns, notsignificant. (D) Dark rearing protects A3C mice from retinal degeneration.Retinal morphology and the ONL thickness of 4-mo-old control and A3Cmice. (Scale bar: 50 μm.) *P = 0.011; **P = 0.006.

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was relatively slow, with the ONL thickness decreasing to ∼29 μmat 7 mo and ∼20 μm at 12 mo (Fig. 2C and Fig. S6). There wereno significant differences in the thickness of the inner nuclearlayer between control and A3C mice, indicating that the down-stream retina remained intact.We determined if retinal degeneration in A3C mice could be

alleviated by rearing animals in darkness. After weaning (P20),A3C mice were maintained in light-protected cabinets until theage of 4 mo, at which point their retinal morphology was ex-amined. Dark rearing seemed to prevent A3C retinas from de-generation, because the ONL thickness of dark-reared A3C miceat 4 mo was ∼46 μm, unlike the ONL thickness of the A3C micemaintained in a normal light/dark cycle (Fig. 2D).

Transducin Translocation Desensitizes Rod Phototransduction. Weexamined the functional consequences of impaired transducintranslocation in mouse rods. Dark-adapted rods of control andA3C mice displayed similar sensitivity, which is in good agree-ment with previous studies (20–22). The rising phases of dim flashresponses were similar in dark-adapted control and A3C rods,with A3C rods displaying a modestly slowed recovery to baseline.An additional membrane restraint of Gαt1A3C by the palmitoylanchor may preclude an optimal interaction and inactivation ofthe Gαt1A3C/PDE6 complex by the regulator of G protein sig-naling 9 (RGS9) GTPase-accelerating protein (GAP) complex.The times to peak of dim flash responses in control and A3C rodswere statistically indistinguishable (Table 1), and the calculatedamplification constant for phototransduction was also statisticallyindistinguishable (Fig. S7) (4, 23), indicating that dark-adaptedphototransduction was relatively normal in both genotypes.After the translocation protocol (Materials and Methods and SI

Text), control mice displayed adapted rod photoresponses withreduced sensitivity and shorter times-to-peak and recovery timeconstants (Fig. 3 and Fig. S7). The amplification constant forphototransduction under these conditions was reduced by fivefoldcompared with darkness, which is similar to the findings in a pre-vious work (4). This reduction in sensitivity can be attributed toboth a lowered gain of signaling caused by reduced transducinpresence in the OS (4) and desensitization as expected for partiallybleached rods (24) (Materials and Methods and SI Text). However,A3C rods displayed little change in sensitivity after the trans-location protocol and were approximately fivefold more sensitivecompared with control rods (Fig. 3). In addition, the A3C rodamplification constant was reduced by 1.8 fold, a lesser extent thanfor control rods (Fig. S7). Furthermore, A3C rods did not displayaccelerated response kinetics in the dim flash response, which wasobserved in control rods (Fig. S7). This lack of desensitizationpresumably results from both an absence of transducin trans-location and perhaps, the inability of free opsin to activate A3C

transducin (Discussion). Unfortunately, biochemical evaluation ofGαt1A3C activation by opsin does not seem feasible. The palmi-toylated transducin was not extracted from the membrane (Fig.1E), and hydroxylamine treatment to produce rod outer segmentmembranes with opsin only would cleave off the palmitoyl moiety.However, the observed changes in dim flash response kinetics andsensitivity after the translocation protocol are consistent with a rolefor transducin translocation in normal rod function and adaptation.

Transducin Translocation Enhances Signal Transmission to Rod BipolarCells. In mammalian retinas, the glutamate release from rods issensed primarily by a subclass of ONbipolar cells called rod bipolarcells (18). Dark-adapted rod bipolar cells of control and A3C micedisplayed similar sensitivity (Fig. 3), which is in good agreementwith previous studies (22, 25). Surprisingly, after the translocationprotocol, there was a relatively minor (∼1.6-fold) reduction insensitivity of control rod bipolar cells from the fully dark-adaptedstate (Table 1). However, A3C rod bipolar cells displayed ap-proximately threefold reduced sensitivity from the dark-adaptedstate, and thus, they were approximately twofold less sensitive thancontrol rod bipolar cells under the same experimental conditions.It should be noted that A3C rods using the translocation protocolwere approximately fivefold more sensitive than control rods.Thus, despite the reduction in the sensitivity of control rods pro-duced by light-induced transducin translocation, control rod bi-polar cells seem to retain greater sensitivity than A3C rod bipolarcells. The exclusive expression of Gαt1 and Gαt1A3C in rods andthe reduced sensitivity of A3C rod bipolar cells together suggestthat, under normal circumstances, light-translocated transducinenhances signal transmission from rods to rod bipolar cells.

Desensitization in A3C Rod Bipolar Cells Is Not Caused by Changes inRod Membrane Properties. The reduced sensitivity of light-adaptedA3C rod bipolar cells, compared with control, indicates that,under normal circumstances, transducin translocation to the rodIS or spherule improves the sensitivity of signal transmission torod bipolar cells. In principle, transducin translocation to rod ISand spherules may alter the functional properties of three classesof targets: (i) transducin may enhance IS voltage-sensitive con-ductances, such as Ih (26), that increase the rod hyperpolariza-tion per absorbed photon, (ii) transducin translocation may alterthe voltage sensitivity of CaV1.4 Ca2+ channels, thereby allowinga larger relative reduction in synaptic Ca2+ per absorbed photon,or (iii) absent an influence of transducin on IS or synaptic Ca2+

conductances, transducin may interact with the glutamate releasemachinery to produce a larger reduction in glutamate release perabsorbed photon. We tested explicitly the first two possibilities todetermine whether they could contribute to enhanced signaltransmission to rod bipolar cells.

Table 1. Response characteristics of control and A3C rods and rod bipolar cells

Imax (pA)* I1/2 (R*/rod) TTP (ms)† or Hill exponent (n) τrec (ms)

RodsControl DA 17 ± 1.6 (5) 10 ± 0.7 (6) 240 ± 15 (507) 220 ± 23 (6)Control LA 15 ± 1.3 (8) 80 ± 2.8 (3) 190 ± 12 (597) 150 ± 11 (3)A3C DA 20 ± 1.5 (7) 11 ± 1.2 (3) 280 ± 15 (986) 410 ± 43 (3)A3C LA 22 ± 0.9 (7) 17 ± 1.3 (5) 280 ± 12 (457) 420 ± 39 (5)

Rod bipolar cellsControl DA 480 ± 60 (6) 2.8 ± 0.3 (6) 1.7 ± 0.1 (6)‡

Control LA 130 ± 31 (7) 4.6 ± 0.3 (7) 1.4 ± 0.1 (7)‡

A3C DA 280 ± 53 (7) 2.3 ± 0.3 (7) 1.6 ± 0.1 (7)‡

A3C LA 90 ± 24 (6) 7.5 ± 0.8 (6) 1.2 ± 0.1 (6)‡

DA, dark-adapted; Imax, light-evoked dark current; I1/2, half-maximal flash strength; LA, light-adapted; n, bestfit Hill equation to the respective intensity–response curves; τrec, time constant describing the exponential thatfits the decay of the dim flash response; TTP, time to peak of the dim flash response.*All results are presented as mean ± SEM (n cells).†TTP for rod dim flash responses are presented as mean ± SEM (number of trials).‡Hill exponent.

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Rod IS currents were measured while varying the membranepotential. As shown in Fig. 4A, rods were held at a restingmembrane potential of Vm = −40 mV and stepped for 200 ms tohyperpolarizing potentials in 5-mV increments to Vm = −80 mV.Such hyperpolarizing steps are representative of variations in therod membrane potential during the light-evoked response (27).In addition, the duration of the voltage step exceeded the normaltime to peak of the downstream rod bipolar cell light-evokedresponse. The current at steady state was plotted against themembrane potential in the dark-adapted rods using the trans-location protocol for control (Fig. 4B, Left) and A3C mice (Fig.4B, Right). We found that, in both control and A3C rods, thecurrents evoked by hyperpolarizing steps in membrane potentialwere statistically indistinguishable. Thus, light-induced transducin

translocation does not seem to significantly influence rod ISconductances that shape the rod’s voltage response.Synaptic transmission from rods to rod bipolar cells can also

be modulated through the tuning of the voltage sensitivity ofCaV1.4 Ca2+ channels (28). The increased sensitivity of controlrod bipolar cells might then be produced by a shift of the voltagesensitivity to hyperpolarized membrane potentials, which wouldenhance Ca2+ influx. We measured the voltage sensitivity of thesynaptic Ca2+ current by ramping the membrane potential ofrods and measuring the resulting current (Materials and Methodsand SI Text). The recording conditions isolate appropriately theCa2+ current, which is blocked completely by the selective in-hibitor isradipine (29) (Fig. 4C). Measurements of the Ca2+current from control or A3C rods in the dark-adapted state orafter light-induced transducin translocation display a similarshape of the voltage dependence (and thus, a similar Ca2+conductance) and no significant shift in voltage sensitivity, es-pecially in the physiological range of rod voltages (Fig. 4D). Inaddition, the Ca2+ charge transfer, taken as the integral of thecurrent response between −80 and −40 mV, was not significantlydifferent in control or A3C mice under dark- or light-adaptedconditions. Thus, light-induced transducin translocation does notseem to influence significantly Ca2+ influx to rod spherules.

DiscussionA feature of synapses is their ability to adjust strength in anactivity-dependent manner. Such regulation is common in thevisual system and responsible for the wide range of light in-tensities that it encodes. Here, we studied the role of transducintranslocation in regulating the functional output of rod photo-receptors. Specifically, based on the diffusion model of light-dependent translocation (10, 12, 13), we have developed atransgenic mouse model where native rod transducin has beenreplaced with a mutant transducin, which displays impairedtranslocation. Analysis of retinal architecture and signal trans-mission in transgenic mice reveals that impaired translocationcaused a slow light-dependent retinal degeneration and alsoimpaired signal transmission to rod bipolar cells.

Additional Lipidation of Transducin-α Hinders Light-EvokedTranslocation. The RAC assay indicated that at least 40% ofGαt1A3C in transgenic rods is palmitoylated. Palmitoylationof Gαt1A3C apparently occurs in the IS, because most proteinacyltransferases (Asp-His-His-Cys protein family) are localizedin the endoplasmic reticulum and Golgi (30). Thus, Gαt1A3C isnot fully palmitoylated in the IS or perhaps, is slowly depalmi-toylated in the OS (31). Gαi and Gαo are largely depalmitoylatedduring purification (32). In view of the labile nature of thethioester linkage of S-acylation, the extent of Gαt1A3C palmi-toylation in intact rods might be greater than the estimate fromthe RAC assay. Palmitoylated Gαt1A3C resisted extraction fromthe membranes with isotonic buffer containing GTPγS, con-firming our prediction that the dual lipidation, N-acylation andS-palmitoylation, would firmly anchor the protein to the mem-brane. Although palmitoylation of Gα subunits has been repor-ted to target G proteins to lipid rafts (33), only a small fraction ofGαt1A3C was found in the Triton-100 insoluble fraction. Pal-mitoylation of Gαt1A3C did not interfere with proper traffickingof transducin, which was localized to the OS in dark-adaptedA3C mice. More importantly, light-dependent transducin trans-location in A3C mice was noticeably impaired, apparently be-cause of tight association of the palmitoylated Gαt1A3C with thedisk membrane (Fig. 2 A and B). Translocations of both Gαt1A3Cand Gβ1γ1 were deficient in mutant mice. The requirement ofGαt1 translocation for efficient redistribution of Gβ1γ1 in light-adapted rods is consistent with the sink model, where Gαt1 andGβ1γ1 accumulate in the IS by forming a heterotrimer in theabsence of R*-dependent activation (12).

Light-Evoked Transducin Translocation Promotes Rod Survival. In-terestingly, A3C mice develop photoreceptor degeneration under

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Fig. 3. Dark- and light-adapted rod and rod bipolar cell responses in controland A3C mice. (A) Representative flash response families from control (Gαt1+/−)and A3C rods in the dark-adapted state and after the translocation protocol.Timing of the 30-ms flash is indicated by the arrowheads, and flash strengthsranged from 0.6 to 540 R*/rod for the dark-adapted state and from 2.3 to 1,700R*/rod for the translocated state. (B) Response–intensity relationships for dark-adapted (DA; black) and translocated (LA; gray) rods from control (Upper) andA3C (Lower) mice. Normalized flash responses were plotted as a function offlash strength. Sensitivity was estimated from the half-maximal flash strengthof the Hill curve indicated by arrows for each state and genotype (Table 1).Arrows are included to indicate the half-maximal flash strength, with a solidline denoting the half-maximal flash strength of DA control and A3C rods,a dashed line denoting the half-maximal flash strength of LA control rods, anda dotted line denoting the half-maximal flash strength of LA A3C rods. (C)Representative flash response families from the different rod bipolar cells.Timing of the 10-ms flash is indicated by arrowheads, and flash strengthsranged from 0.3 to 21 R*/rod for the dark-adapted state and from 1.0 to 70 R*/rod for the light-adapted, translocated state. (D) Response–intensity relation-ships for dark-adapted (DA; black) and translocated (LA; gray) rod bipolar cellsfrom control (Upper) and A3C (Lower) mice. Normalized flash responses wereplotted as a function of flash strength. Sensitivity was estimated from the half-maximal flash strength of the Hill curve, which is indicated by arrows for eachstate and genotype (Table 1). Arrows are also included to indicate the half-maximal flash strength for comparison with rods. Solid lines denote the half-maximal flash strength of DA control and A3C rod bipolar cells, a dashed linedenotes the half-maximal flash strength of LA control rod bipolar cells, anda dotted line denotes the half-maximal flash strength of LA A3C rod bipolarcells. Note the reversed position of the dashed and dotted lines in B and D. Allrecords were sampled at 1 kHz and low pass-filtered at 50 Hz.

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conditions of a 12/12-h light/dark cycle with modest light in-tensities of ∼100–200 lx (Fig. 2C and Fig. S6). These lightconditions, nonetheless, are sufficient to trigger transducintranslocation in WT rods (6, 15, 34). Dark rearing protected A3Cmice from retinal degeneration, indicating that this photore-ceptor death is light-dependent. Our results suggest two plausi-ble reasons for the retinal degeneration, both of which lead topersistent high phototransduction gain in A3C rods. First,normal light-induced transducin translocation correlates with areduction in the gain of phototransduction (4). Therefore,preventing light-induced transducin translocation would beexpected to maintain a high gain of phototransduction, a hall-mark of dark-adapted rods. Second, our recordings of A3C rodphotocurrents reveal that little desensitization/adaptation isobserved using the translocation protocol, which leads to a re-sidual 35% bleached visual pigment (Materials and Methods andSI Text). It is well known that, after visual pigment bleachingin both amphibian and mammalian rods, the sensitivity of pho-totransduction is reduced because of the sum of the loss ofquantum catch and residual phototransduction activity producedby free opsin (24, 35). The relatively minor desensitizationof A3C rods using the translocation protocol (Fig. 3B) can beexplained by the loss of quantum catch alone and thus, sug-gests that free opsin is unable to activate A3C transducin. Thispresumed lack of bleaching adaptation would also prevent a re-duction in phototransduction gain. Taken together, the lack oftransducin translocation and lack of bleaching adaptation wouldcause the overactivation of phototransduction in bright light andperhaps, the resulting retinal degeneration (8).

Light-Evoked Transducin Translocation Enhances Signal Transmissionfrom Rods to Rod Bipolar Cells. The dark-adapted sensitivities ofboth rods and rod bipolar cells were similar in control and A3Cmice, which was expected based on the similar concentration ofthe G protein in both strains and the similar ability of Gαt1 andGαt1A3C to be activated by R*. However, using the translocationprotocol, we find substantial differences between control and A3Cmice. The rods of A3C mice retained fivefold greater sensitivityunder these conditions compared with control rods, but the A3C

rod bipolar cells displayed twofold reduced sensitivity comparedwith control rod bipolar cells. This reversal indicates that the light-induced translocation of transducin has an influence downstreamof the rod OS to increase the sensitivity of signal transmission.Given the specific expression of Gαt1 and Gαt1A3C in rod

photoreceptors and given that the dark-adapted sensitivity ofcontrol and A3C rods and rod bipolar cells is indistinguishable(Fig. 3), the lack of a major influence of Gαt on IS and synapticcurrents (Fig. 4) is suggestive of a presynaptic interaction oftransducin with the synaptic machinery. Although the exact natureof this interaction remains unclear, there is considerable evidencethat shows G protein-coupled receptor signaling actively regulatesof synaptic transmission in the CNS (36). Both Gαt1 and Gβ1γ1translocate to the rod spherule in bright light, where they mayform a heterotrimer. However, dissociated Gαt1 and Gβ1γ1 speciescan be generated in the spherule through the Gαt1 interaction withits protein partner UNC119 (37–39). Thus, in principle, either ofthese proteins may mediate the observed effect on signal trans-mission. For example, Gβ1γ1 has been shown to inhibit neuro-transmitter release through direct interactions with the SNAREcomplex (40). Specifically, Gβ1γ1 and synaptotagmin have beenshown to compete for binding to SNAP25, syntaxin1A, and theternary SNARE complex in a Ca2+-dependent manner. TheSNARE complexes in central and ribbon synapses are thought tocomprise of homologous proteins (41). Thus, the light-induceddecrease in intracellular Ca2+ in the rod spherule might augmentthe inhibitory action of Gβ1γ1 on the SNARE proteins and facil-itate the reduction in glutamate release. An alternative mecha-nism for transducin’s synaptic sensitization may involve a directaction of Gαt1 with UNC119, a partner for the synaptic ribbonprotein RIBEYE (42). The exact mechanisms of transducinmodulation of rod-to-rod bipolar cell signal transmission willrequire additional investigation.Thus, the light-dependent translocation of transducin in rods

seems to provide adaptive changes to the rod photoresponse tofacilitate light adaptation, which is not seen in A3C mice. Sucha mechanism might protect rods from excessive stimulation underconditions where there is substantial free opsin in the OS. Addi-tionally, transducin-dependent sensitization of signal transmission

A

B

C

D

Fig. 4. Dark- and light-adapted inner segment andsynaptic conductances of control and A3C rods. (A) Rodswere held at Vm = −40 mV and stepped for 200 msto hyperpolarizing potentials in 5-mV increments up toVm=−80mV as shown inUpper. Representative voltagestep families from rods of dark-adapted (black) andtranslocated (gray) control and A3C mice, respectively,are shown. Currents were sampled at 10 kHz and lowpass-filtered at 300 Hz. (B) The measured inner segmentmembrane current is plotted as a function of the mem-brane potential for control and A3C rods. The controlplot is an average of six dark- (black) and nine light-adapted (gray) rods. TheA3Cplot is anaverageof9dark-(black) and10 light-adapted (gray) rods.Dataareplottedas the mean ± SEM. (C) The Ca2+ current was measuredby ramping the rod’s membrane potential between −80and+40mVover1,000ms. The synaptic Ca2+currentwasisolated while blocking other membrane conductanceswith cesium-containing electrode internal solution (SIText). The leak-subtracted Ca2+ current is an averagefrom five cells. The Ca2+ current was blocked totally by10 μM isradipine. (D) Leak-subtracted Ca2+ currents areplotted from dark-adapted (black) and translocated(gray) control and A3C rods. The control plot is an aver-age of three dark- (black) and five light-adapted (gray)rods. The A3C plot is an average offive dark- (black) andfive light-adapted (gray) rods. Data are plotted as themean with 1 SD boundary. Calculations of the Ca2+ cur-rent density in the rod physiological voltage range (−80to −40 mV) were 250 ± 78 pA2 in dark-adapted controlrods (mean ± SEM, n = 3), 180 ± 36 pA2 in light-adaptedcontrol rods (mean ± SEM, n = 4), 170 ± 62 pA2 in dark-adapted A3C rods (mean ± SEM, n = 5), and 200 ± 33 pA2 in light-adapted A3C rods (mean ± SEM, n = 4).

12472 | www.pnas.org/cgi/doi/10.1073/pnas.1222666110 Majumder et al.

may, in turn, allow a larger part of the dynamic range of rod bi-polar cells to be used under conditions where the rod photocur-rent would be strongly desensitized. These direct demonstrationsshow the role of transducin translocation in the adaptation of rodsand rod bipolar cells to background light.

Materials and MethodsTransgenic Mice. Transgenicmicewith impaired transducin translocationweregenerated through the inclusion of an additional posttranslational modifi-cation to Gαt1. A site-directed mutation was made to transducin-α (Fig. 1A),which created the N-terminal S-palmitoylation site akin to the site present inGαi/Gαo (43). The procedures used to generate A3C mice are well-establishedand described fully in SI Text.

Immunohistochemistry and Biochemistry. The procedures used for immuno-blotting, immunofluorescence and retina morphology analyses, transducinextraction, G-protein palmitoylation, and rhodopsin regeneration assays arewell-established and described fully in SI Text.

Physiological Recording from Rods and Rod Bipolar Cells. Rod photocurrentswere measured using suction electrodes, and rod bipolar cell currents weremeasured using patch electrodes and methods described previously (SI Text).Voltage-dependent currents and the voltage sensitivity of Ca2+ currents in rodswere measured with patch clamp electrodes from the rod inner segment inretinal slices. Experiments to measure voltage-dependent currents (Fig. 4 A andB) were performed using a K-Aspartate internal solution (SI Text), but themeasurements of the voltage sensitivity of the synaptic Ca2+ current (Fig. 4 C and

D) were made using a pipette internal solution that blocked other cationiccurrents (SI Text).

Light-Induced Transducin Translocation Protocols. All experiments were per-formed on mice dark-adapted overnight or mice that, after dark adaptation,were exposed to light bright enough to cause transducin translocation. Inbiochemical and immunohistological experiments, dark-adapted mice weresubjected to 500 lx light for 45 min after eye dilation (1% tropicamide and2.5% phenylephrine hydrochloride) and euthanized according to a protocolapproved by the Institutional Animal Care and Use Committee of the Uni-versity of Iowa (Protocol 1005089).

In physiological experiments, the 500 lx light exposure was followed by a 30-min period of dark adaptation to ensure some visual pigment regeneration andrecovery of visual sensitivity. This light exposure variation is also called thetranslocation protocol, and the preparation is referred to as light-adapted. Thevisual pigment regenerated to ∼65% of its dark-adapted level under theseconditions (SI Text). Recordings were always halted at 1 h using the translocationprotocol to ensure that Gαt remained substantially in the rod IS during therecordings (4). Mice used for physiological recordings were between 2 and 3 moof age (before the onset of rod degeneration). Mice were euthanized in ac-cordance with protocols approved by the Institutional Animal Care and UseCommittee of the University of Southern California (Protocol 10890).

ACKNOWLEDGMENTS. We thank Dr. Amy Lee for helpful comments on themanuscript. We also thank Dr. S. Thompson for providing access to custom-built light-protected cabinets for dark rearing of mice. This work wassupported by National Institutes of Health Grants EY17035 (to V.R.),EY17606 (to A.P.S.), and EY12682 (to N.O.A.) and the McKnight EndowmentFund for Neuroscience (A.P.S.).

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