Molecular Vision 2005; 11:1246-1256 <>
Received 18 August 2005 | Accepted 5 December 2005 | Published 31 December 2005

Altered light responses of single rod photoreceptors in transgenic pigs expressing P347L or P347S rhodopsin

Timothy W. Kraft,1,2 Derron E. Allen,1 Robert M. Petters,3 Ying Hao,4 You-Wei Peng,4 Fulton Wong4,5

Departments of 1Physiological Optics and 2Neurobiology, School of Optometry, University of Alabama, Birmingham, AL; 3Department of Animal Science, North Carolina State University, Raleigh, NC; Departments of 4Ophthalmology and 5Neurobiology Duke University Medical Center, Durham, NC

Correspondence to: Timothy W. Kraft, PhD, UAB School of Optometry, WORB 612, 924 18th Street South, Birmingham, AL 35294-4390; Phone: (205) 975-2885; FAX: (205) 934-5725; email:


Purpose: Numerous mutations of rhodopsin lead to rod cell death and ultimately to complete blindness, yet little is known about the alterations in the physiology of the light sensors containing the aberrant protein, the rod photoreceptors.

Methods: Suction pipettes were used to record the light responses from single rod photoreceptors isolated from the retinas of transgenic pigs of various ages and at progressive stages of retinal degeneration.

Results: We have observed changes in the photoresponse of transgenic porcine rods containing both wild type and mutant rhodospin. Our findings are consistent with the idea that substitutions at position proline 347 of rhodopsin interfere with the inactivation of R*. In addition the level of photoreceptor degeneration is positively correlated with an acceleration and desensitization of the photoresponse to dim flashes.

Conclusions: It appears that the phototransduction cascade, even when initiated by wild type rhodopsin molecules is altered in a way that is progressive with the level of retinal degeneration. A model consistent with our observations introduces the idea of a binding site for the carboxy terminus of rhodopsin on rhodopsin kinase.


Close to 100 mutations in the rhodopsin gene have been identified in patients with hereditary retinal degeneration called retinitis pigmentosa (RP) [1-7]. A common feature of the RP phenotypes induced by a large spectrum of mutations, of which rhodopsin is only one of the many genes involved, is early night blindness due to death of rod photoreceptors and protracted degeneration of cone photoreceptors [7,8]. Naturally occurring and transgenic animal models of rhodopsin mutations have been and are being intensely studied [9-18]. These animal models are critical for studying disease mechanisms and for therapeutic studies. Furthermore, correlating alterations in the physiological responses recorded from single rods with the known mutations in rhodopsin will help to elucidate the role of normal and mutant rhodopsin in phototransduction [13,18-20]. A number of the known mutations of rhodopsin that cause retinal degeneration are found in the carboxy terminus, a region of the protein important for the inactivation of photoactivated rhodopsin (R*) and a recognition site for rhodopsin transport [19,21-23]. Mutations removing phosphorylation sites in the carboxy terminus show prolonged photoresponses consistent with perturbations of the R* inactivation pathway [18,20].

Mutant rhodopsin transgenic pigs have been created as models for RP research; the mutations are P347L and P347S, which cause autosomal dominant retinitis pigmentosa (ADRP) in humans. Previous studies have defined the morphological, electroretinographic (ERG), and synaptic changes observed in the P347L pigs [24-29]. Herein we report the alterations in light responses of single rods of P347L and P347S transgenic pigs. Our results are consistent with the notion that, along with normal rhodopsin, the mutant rhodopsin molecules are produced, transported to the outer segment, and participate in phototransduction. However, at the single-photon level, the mutant rhodopsin-initiated light response is substantially different kinetically than those attributed to normal rhodopsin. Summation of a mixture of responses due to activation of normal and mutant rhodopsin may account for the changes in light sensitivity and kinetics of the rod light response observed in the transgenic pigs.


Production and identification of animal genotypes

The production of the transgenic lines used in this study was described elsewhere [26]. In brief, a DNA fragment containing the pig rhodopsin gene mutated in codon 347, CCA(Pro) to CTA (Leu) or TCA (Ser), was microinjected into porcine embryos. Transgenic lines (Pro347Leu and Pro347Ser) were established following Southern hybridization and test matings. Both lines inherited their transgene as a Mendelian trait. Pigs for this study were produced under the supervision of Dr. Petters at North Carolina State University (Raleigh, NC). For the present study, transgenic pigs were identified using PCR. Briefly, the PCR primers PIG 1F (5'-ACT GGG TGA TGA CGA AGG C-3') and PIG 1R (3'-GGC GTG GAC AGT CTT GGT-5'), were used to amplify a 170 bp fragment that was cut with either NlaIV (for Pro347Leu) or HgaI (for Pro347Ser). At three weeks of age the pigs were genotyped and then shipped by air carrier to the University of Alabama at Birmingham (Birmingham, AL).

Determination of transgenic expression

The ratio of mutant to normal RNA was used to estimate the level of expression of the transgene as described previously [26]. Briefly, total retinal RNA samples were extracted from porcine retinas using a guanidinium thiocyanate procedure. Reverse transcription of total RNA was performed; the resulting cDNA, which contained a mixture of fragments derived from normal and mutant rhodopsin RNA, was subjected to PCR amplification using a primer pair that spanned intron 4 of the rhodopsin gene and included codon 347. The PCR products were cloned. Randomly picked single clones were identified as normal or mutant according to the sequence of codon 347. For P347L pigs, mutant RNA was estimated to be 62% [26]. The percentage of P347S RNA in that transgenic line was measured by methods identical to those used for P347L. The result for the P347S line was 58% mutant RNA, based on over 70 samples taken from three pigs, ages 4 weeks, 8 weeks, 13 months.

Tissue preparation and suction electrode recording

All experimental protocols were approved by UAB's institutional animal care and use committee. Animals were anesthetized with intramuscular injections of xylazine and ketamine, then dark-adapted for 45 min. Enucleations were performed under heavy pentobarbitol anesthesia (IV) in complete darkness with the aid of infrared to visible image converters. Animals were sacrificed with a pentobarbital overdose after bilateral enucleation. Details of the tissue preparations and electrolytes have been given previously [30,31]. Briefly, the retina was isolated under infrared light in Lebovitz's L-15 medium and stored at 4 °C in the dark. Experiments were performed on the same retina for four to five days, each tissue sample lasted 3 to 4 h. For each experiment a small piece of a retina (about 3 mm x 4 mm) was removed from cold storage and chopped under infrared light to produce small pieces of retina about 50 to 100 μm on a side, then warmed to near body temperature in a perfusion chamber with a bicarbonate buffer that contained the following: 120 mM NaCl, 20 mM NaHCO3, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 3 mM HEPES, 0.02 mM EDTA, 10 mM D-glucose, plus Gibco's MEM vitamin mixture. The circulating dark current of individual rods or cones was recorded by drawing the outer segment into a suction electrode whose inner diameter matched the outer segment diameter of the cell [32]. The suction electrode was filled with a buffer solution that contained the following in mM: NaCl (140), KCl (3.6), MgCl2 (2.4), CaCl2 (1.2), HEPES (3), EDTA (0.02) D-glucose (10), pH 7.4. The photocurrent and stimulus-monitor signals were typically digitized at 2 ms intervals with hardware (MIO16) and software (LabView) from National Instruments (Austin, TX). A stimulus set consisted of 5 to 30 responses to the same wavelength and intensity of light. The light bench focused a 440 μm diameter spot of light at the plane of the cells. The wavelength was controlled with a 3-cavity interference filter (Andover, NH) with a peak transmission of 501.7 nm and a bandwidth of 10 nm. Neutral density filters (Reynard, CA) attenuated the light. Calibration of unattenuated light was performed daily with a photometer (Graseby Optronics, FL; Model 350).


Cell marker-specific antibodies were anti-rhodopsin mouse monoclonal antibodies (a gift from P. Hargrave, University of Florida, Gainesville, FL) and anti-cone transducin g rabbit polyclonal antibodies (CytoSignal, Irvine, CA). Retinal sections, 6 to 8 μm thick, were incubated with 5% normal goat serum (Vector Laboratories) in phosphate-buffer saline (PBS) for 1 h at room temperature, and then incubated with primary antibodies (anti-rhodopsin antibody diluted 1:2000 and anti-transducin g antibody 1:1000) overnight at 4 °C followed by three washes in PBS for 15 min. each. All incubation and wash buffers contained Triton X-100 (0.3%). The sections were then incubated with either rhodamine-conjugated goat anti-mouse immunoglobulin antibody 1:50 or, with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin antibody 1:50 for 2 h at room temperature. After three more washes with PBS for 15 min each, the slides were then coverslipped with 50% glycerol in PBS for viewing under a Zeiss microscope.


The photocurrent responses recorded from wild type porcine rods are qualitatively very similar to rod responses recorded from other mammalian species (Figure 1A) [19,31,33,34]. Although the time to the peak of the response is slower than other mammals, we verified that the massed photoreceptor response matched the kinetics of the suction electrode recordings of individual cells using sheets of retinal tissue and an Ussing Chamber to record the fast PIII (data not shown). Rods from transgenic animals gave responses that were different in several respects from the wild type rod responses. The time-to-peak of linear and mid-range responses was greatly accelerated, the recovery phase of the responses was delayed, and the sensitivity of the cells to light was reduced. The P347L rods had a small but consistent delay in the recovery; note the persistent photocurrent remaining one second after the flash (Figure 1B). The five P347L animals used ranged in age from 4.5 to 10.5 weeks old at the time of sacrifice. Table 1 gives response and sensitivity parameters for a collection of rods from five wild type and five P347L animals. The results from the wild type and P347L animals were very consistent and have been pooled. The time-to-peak of the P347L rods was reduced 2.1 fold, compared to wild type rods containing only normal rhodopsin. The sensitivity of the P347L rods was reduced by 6.7 fold based on the flash sensitivity (Sf). The integration time of the P347L rods is reduced by exactly the same factor as the time-to-peak. Calculations of the phototransduction gain using the rising phase of the family of light responses like those in Figure 1A resulted in similar values of gain for all three genotypes.

Rods containing P347S rhodopsin also produced responses with a very rapid time-to-peak with a response recovery that was profoundly delayed (Figure 1C). Results from the P347S rods were variable from animal to animal and therefore were considered only within each animal [25]. Results for five P347S animals with different levels of degeneration are given in Table 2. Two animals of similar age are identified as "P347S fast" for fast degeneration and fast time-to-peak (Table 2, row 3) and "P347S slow" for slow degeneration and slow time-to-peak (Table 2, row 5). Figure 1C shows a family of responses from the P347S fast animal.

The stimulus intensity versus response-amplitude data for the three cells in Figure 1A-C are plotted in Figure 1D. Response amplitudes were normalized for each cell and the data were fit with an exponential saturation function of the type used previously to describe rod and cone response behavior [35]. The open squares and filled triangles representing the transgenic rods are shifted to the right indicating an approximately fivefold loss of sensitivity in these cells. On average, the P347L-containing rods were 3.2 fold less sensitive than wild type rods, based on I1/2, the light intensity required to evoke a half-maximal response (Table 1). Based on I1/2, rods from the "P347S fast" animal (Figure 1C, dashed line) were desensitized 3.7 fold (Table 2, row 3). In contrast, rods from the slowly degenerating P347S animal ("P347S Slow") were desensitized only 1.6 fold (Figure 1F, light gray line; Table 2 row 5).

When midrange responses (33% to 42% of the cells' maxima) were scaled to match their peak amplitudes, the kinetic differences between wild type and P347L transgenic rods (Figure 1E) and wild type and P347S rods (Figure 1F) were apparent. The rapid rising phase was due to the insensitivity of the transgenic rods and the greater stimulus strength required to generate these responses (Figure 1D, Table 1). An analysis of the phototransduction gain after Lamb and Pugh [36] showed no difference in the amplification constant (A) between the wild type and transgenic rods. Thus, the faster time-to-peak indicates that the shut-off mechanisms that generate the peak response are somehow stimulated or enhanced in these cells. However, the slow to very slow final recovery suggests that some component of the photocurrent-generating cascade is resistant to inactivation. These two seemingly contradictory observations regarding the shutoff mechanisms can be further dissected by looking carefully at the single-photon events evoked by very dim flashes, and will be discussed below. Figure 1F shows three responses: The solid black line is from the wild type rod and the other two traces are from rods of P347S animals. Both transgenic rods show the very slow recovery component, but one has greatly accelerated time-to-peak ("P347S fast", dashed line) while the other has a rising phase and peak time rather similar to wild type rods ("P347S slow", light gray line). The faster time to peak was observed in the cells from an animal with more advanced degeneration.

The variability observed in the kinetics (time-to-peak) of the rod responses recorded from the P347S animals follows a pattern that is demonstrated when the level of retinal degeneration (rod loss) is considered. Degeneration level was quantified in retinas from five P347S animals by identifying rods with an anti-rhodopsin antibody and then counting the number of rod nuclei present. In wild type rods, rhodopsin localized to only the outer segments (Figure 2A, left image). In P347S pigs, rhodopsin was distributed in outer segments, inner segments, and synaptic membranes (Figure 2A, right image) [24]. In the region of the cell bodies, the cell nuclei were identified for counting by a ring of antibody staining signal surrounding each rod nucleus. Figure 2B shows the direct relationship between accelerated time-to-peak and decreasing rod count (left axis). The time-to-peak was halved as the rod count declined by a factor of 10. The sensitivity parameters of the cells (I1/2, flash sensitivity) followed a similar pattern (Table 2). The cone densities were unaffected, even in the presence of massive rod loss (Figure 2B, circles, right axis). Ultimately the cones in this model of RP will die [26], but their numbers had not yet begun to decline at the time of our recordings when up to 90% of rods were gone.

The dramatic differences in the kinetics of the very dim flash responses that accompany a twofold decrease in the time-to-peak are shown in Figure 2C. The response from a wild type rod peaks at 310 ms, while the average of the sorted singles of a rod from a degenerating retina (see dim flash analysis below) peaks at 165 ms and has recovered almost 40% by 310 ms.

Dim flash analysis

Rods are single-photon detectors capable of demonstrating the quantal nature of light. Their responses to weak stimuli have been shown to follow a Poisson distribution [37]. Thus, by using weak stimuli that on average produce less than a single photoisomerized rhodopsin molecule per flash, we can stimulate either a wild type or mutant rhodopsin independently within a single cell. Previous work with other types of rhodopsin mutations or phototransduction-protein mutations has revealed distinct single-photon-like events suggestive of underlying changes in the phototransduction cascade [18,20,38-43]. The responses of rods containing P347L and P347S rhodopsin to a series of very weak stimuli revealed two classes of light response (in addition to "blank" responses frequently observed when, due to the low stimulus intensity, no photon was absorbed by the rod (Figure 3A, bottom trace)). The first class of light response was a stereotypical single-photon response, like those classically recorded in rods (labeled "Singles" in Figure 3A, middle trace). The second class of light response was step-like, rising along a similar time course as the singles, but reaching a greater amplitude and plateaued for a variable period of time, sometimes lasting longer than one second (Figure 3A, upper trace). These step-like photocurrents, or "Steps", were of variable duration, and shut off with an abrupt recovery to baseline, similar to the rising phases. If the lifetime of the step currents was controlled by a reaction that followed first order rate kinetics, such as a single molecular event, then the lifetimes of the step currents should be exponentially distributed. Averaging many step currents reduced the noise giving a clear view of the rising phase and plateau period of the response (Figure 3B, upper trace). The slow recovery to baseline matches the slow recovery seen in the photocurrents produced by bright light flashes, and is well fit by a single exponential. The average of the singles looks similar to the dim flash response from a wild type rod, but with faster overall kinetics (Figure 3B, lower trace). The time to the peak of the averaged "singles" response in P347L rods was about twofold faster than that of the wild type dim flash response (Figure 2C). Ambiguous or intermediate waveforms were omitted; these were usually less than 5% of records.

The light responses of the rods containing P347S rhodopsin had a more protracted recovery phase. Averages of the "singles" and "steps" from dim flash responses in a P347S rod demonstrated the two classes of light response (Figure 4). The average of the singles resembled the form of the dim flash response in wild type rods (Figure 4A, lower trace), except that the kinetics of the responses was accelerated. The step currents observed in P347S rods rose to a similar plateau level as seen in the P347L recordings, but the duration of the plateau was extended and the recovery phase of the photocurrent had a slower time course. For this cell, the duration of each step-current was measured as its width at half-maximum amplitude and was highly variable, mean 1.67±0.77 s (n=26), compared with the width of the singles, mean 0.47±0.10 s (n=10). The recovery time constant for the averaged step response was 643 ms in this cell and averaged 459 ms in 16 cells from P347S animals. Similar dim flash experiments on wild type rods revealed large numbers of blank and single responses and only occasionally the step responses, but step like responses are know to occur with low frequency in wild type rods [30].

A demonstration of the quantal nature of the step responses is given by the histogram of response amplitudes measured over the typical plateau period of the mean step response. Figure 4A shows the mean photocurrent response to 29 flashes that produced a step response (upper trace), and 21 flashes judged to have produced the classical wild type single-photon response (lower trace). The histogram in Figure 4B gives the amplitude distribution for the each of the 29 step responses over the time period indicated by the black bar (from 0.38 to 1.17 s after the flash). The smooth curve is the fit of the sum of three Gaussian curves to the histogram. The peaks of the Gaussians occur at 1.1, 2.2 and 2.8 pA. The histogram of the response amplitudes of the 21 single responses over the central 0.12 s (indicated by the black bar in Figure 4A, lower trace) is well fit by a single Gaussian centered at 0.7 pA amplitude (Figure 4C).

The three peaks of the Gaussian fit to the step response amplitude histogram suggest a quantal distribution of amplitudes (Figure 4B). We tested this idea quantitatively by using a criterion amplitude of 1.5 pA, indicated by the arrow, to sort the individual step responses into quantal events of one, two, or more photon absorptions. Observing a Poisson distribution would support the idea that steps are quantal events produced by single-photon absorption. The 29 steps sorted into 19 single photon events, 7 doubles, 2 triples, and one blank or zero photon event. The Poisson distribution predicts 18.1, 7.4, and 2.0 of the single, double, and triple quantal events in strong support of the notion that step currents are quantized (Table 3).

The rods of these transgenic animals express both the mutant and wild type rhodopsin proteins in the outer segment. If we assume that the mutant rhodopsin causes the step-like responses and "singles" are the photoresponses generated by the activation of a wild type rhodopsin, then from the frequencies of the two types of photoresponses and the Poisson distribution we can estimate the proportion of each type of rhodopsin in the outer segment. Table 3 shows that a 60/40 split of mutant to wild type rhodopsin predicts the observed proportion of steps and singles in this cell. Similar analysis in a number of rods from different animals gave results ranging from 59% to 74% mutant protein (mean= 67±5.6%, n=10). In P347S pigs, the mutation was observed in 59% (10 of 17) and in 62% (21 of 34) of the PCR fragments in a four-week-old and an eight-week-old animal, respectively, and slightly lower (52%) in sample from a 13-month old animal. From these results, the mutant RNA was estimated to be 58% of total rhodopsin RNA. Thus, the functional rhodopsin in the outer segment appears to have proportions of mutant and wild type rhodopsin similar to that of the mRNA suggesting that the mutant rhodopsin does get into the outer segment disks and the plasma membrane.

All of our observations are consistent with the idea that the mutation of the proline residue at position 347 interferes with R* inactivation. A model that can account for these observations is presented in Figure 5. The essential components of the model are a binding site for the C-terminus of rhodopsin on rhodopsin kinase, and that the proline residue at position 347 produces a "hook" at the C-terminus critical to the interaction with this binding site on RK. With wild type rhodopsin the hook tethers the free end of the C-terminus to RK greatly increasing the probability of interaction with the catalytic site of RK. Thus, phosphorylation and R* inactivation of wild type rhodopsin occur quickly and with little variability. Mutations that alter the proline hook result in poor or ineffective binding of the C-terminus and highly variable periods of time between the multiple phosphorylations necessary to inactivate rhodopsin resulting in the prolonged activation state demonstrated by the step currents. Thus, the model predicts that there is a binding site for the terminal five amino acids of rhodopsin somewhere on rhodopsin kinase, and that proline 347 is a key element in creating the structure that is recognized by that binding site.


The light responses from rods isolated from wild type pig are similar to those generated by rods in other mammals. However the porcine rod responses observed had a slower time to peak than those of rodents or primates. The rods from both P347L and P347S transgenic pigs had two altered properties as demonstrated by the photocurrent. The first is a protracted recovery phase that we believe is due to the presence of the ADRP-causing mutant rhodopsin present in the outer segment disks. The second change is a progressive acceleration of the time-to-peak of the response accompanied by a loss of sensitivity. The photocurrent amplitudes measured in wild type and P347S containing rods was similar, but those found in P347L rods were roughly halved. However the decrease in sensitivity observed in the rods from transgenic pigs, when present, was found across all cells independent of the magnitude of the photocurrent.

Several rodents lines with naturally occurring or transgenic alterations of phototransduction proteins have been investigated, most of which also show a delay in the recovery phase of the light response. RGS9 knockout mice have rods with reduced recovery rates and increased overall amplification of the single-photon responses of rods [40]. In the GCAP knockout mouse the single-photon response was five times larger and the time to peak three fold slower [44] due to an interruption of the Ca2+ feedback to guanylate cyclase. However, those animals have a delayed recovery and increased gain, whereas we observe slowed recovery and desensitization in the ADRP-like porcine transgenic animals. Transgenic mice with the point mutation of PDEγ, (W70A), have very insensitive rods with two very distinct phases of recovery, the second of which was sevenfold slower than controls suggesting that interaction with PDEγis important for GTPase activity and inactivation of PDE* [43]. Arrestin knockout experiments showed a normal rising phase and initial recovery, but the final phase of the recovery was dramatically delayed [38]. With the GRK1 knockout mouse the quantal events in rods were greater in amplitude and had an average lifetime of over 3 s [41]. A rhodopsin kinase null mutation in man also slows recovery kinetics [39]. Mutations of rhodopsin in which phosphorylation sites were substituted [18] or deleted [20] delayed the recovery phase of the rod response. Truncation of rhodopsin eliminating all phosphorylation sites produced rods with a mixture of quantal events, some normal, some prolonged, just like those we observed in our porcine model. The prolonged photoresponses of these cells had an average lifetime of almost 6 s. Truncation of just the last four amino acids of rhodopsin (Q344ter) resulted in a 15% increase in time to peak and a 68% increase in flash sensitivity [19], even when only 44% of expression is due to the transgene. Thus, the phototransduction cascade in rods is delicately balanced and perturbations almost always result in delays in the recovery of the light response, which itself could be a triggering event causing retinal degeneration.

Lem and colleagues examined the effects of a rhodopsin knockout and the more interesting electrophysiological properties of the knockout hemizygote with 50% of normal rhodopsin content [33]. The dim flash response of these rods revealed an accelerated time to peak and decreased sensitivity. The retinas of these animals degenerated slowly, but the authors attributed the changes they observed in the flash response to an alleviation of crowding on the disc membrane [45].

We propose that the two classes of single-photon responses observed in P347S rods are generated by the isomerization of either a normal or mutant rhodopsin molecule. Thus, the mutant rhodopsin is synthesized and transported in some way to the outer segment and participates in phototransduction. It has previously been argued that the five terminal amino acids of rhodopsin are a critical recognition factor for the proper sorting of rhodopsin to the outersegment [19,21-23,46]. In earlier studies on transgenic mice, rhodopsin antibodies revealed that P347S rhodopsin is mislocalized to the plasma membrane of the inner segments and synaptic region [23]. P347S and P347L rhodopsin are each mislocalized to the plasma membrane in transgenic pigs, but our results point out that these mutant rhodopsin molecules are present and functional within the outersegment. In addition mutations away from the carboxy terminus of rhodopsin also may result in abnormal placement of rhodopsin [9].

Our interpretation of the step responses and "single" responses sorted from the dim flash results is that the mutant rhodopsin produces the step responses, and that wild type rhodopsin produces a wild type single-photon response. In the slowly degenerating or very young P347S animals (Figure 4A, Table 2) this is just what is seen. In the P347L animals and faster degenerating P347S animals the singles have accelerated kinetics suggesting a reduction in the phototransduction gain or an up-regulation of the inactivation steps of transduction. The graded changes in kinetics seen with graded levels of degeneration in Figure 2B, suggests that whatever retinal changes are induced, they are induced gradually. The consistency of the P347L results suggests that the rapid degeneration that has occurred in these animals has produced a maximal state of adaptation in the surviving rods tested. Whereas the P347S genotype produces a slower degeneration, one in which the physiological state of the retina and thus the state of the rods surviving within the retina change more gradually.

The accelerated time-to-peak and desensitization observed in photoresponses of rods from transgenic animals are consistent with strong light-adaptation. However mammalian rods have shown little or no adaptation [34,47,48], and very small changes in the time-to-peak of the response, even with dramatic changes in sensitivity [49,50]. Perhaps there are regulatory mechanisms that, in the face of consistently prolonged light responses, will induce the up-regulation of proteins responsible for the shut-off of the phototransduction cascade. The retinal degeneration associated with RP induces neurite sprouting in human retina [51] and new synapse formation in pig retina [28], thus it is likely that a host of genes undergo changes in their regulation.

Our observations indicate that the proline in position 347 is somehow important in regulating the shut-off of activated rhodopsin. The model in Figure 5 suggests that proline 347 is part of a binding element or "hook" which attaches to rhodopsin kinase restricting the mobility of the carboxy terminus, thus increasing the probability of the phosphorylation sites interacting with the catalytic sites on rhodopsin kinase. This hypothesis is consistent with the finding that eGFP-rhodopsin fusion protein is poorly phosphorylated when the phosphorylation sites are displace away from the cytoplasmic surface of rhodopsin [52,53] and that phosphorlyation patterns are altered by the absence of the terminal four amino acids of rhodopsin [54]. An alternate mechanism or possibly additional complication, causing a delay in the inactivation of rhodospin would be altered interactions with arrestin. Ohguro [55] found that P347L and P347S carboxy terminal fragments are phosphorylated by RK, but in a different pattern than phosphorylation of wild type rhodospin and, importantly the rates of these phosphorylation reactions are not known. The pattern of C-terminal phophorylation is likely to be critical to proper recognition by arrestin [56-58] and thus delays in the final inactivation of R*, of the order of hundreds of milliseconds that we observe, could result from altered rhodopsin-arrestin interactions.


The authors thank Denis Baylor for his critical review of the manuscript and Jerry Millican for technical assistance, and acknowledge support from NEI grants EY10573 (TWK), and EY11498 (FW), and P30EY05722 (FW), and the Foundation Fighting Blindness and Research to Prevent Blindness (FW).


1. Gal A, Apfelstedt-Sylla E, Janecke AR, Zrenner E. Rhodopsin mutations in inherited retinal dystrophies and dysfunctions. Prog Retin Eye Res 1997; 16:51-79.

2. Jacobson SG, Kemp CM, Cideciyan AV, Macke JP, Sung CH, Nathans J. Phenotypes of stop codon and splice site rhodopsin mutations causing retinitis pigmentosa. Invest Ophthalmol Vis Sci 1994; 35:2521-34.

3. Sung CH, Davenport CM, Nathans J. Rhodopsin mutations responsible for autosomal dominant retinitis pigmentosa. Clustering of functional classes along the polypeptide chain. J Biol Chem 1993; 268:26645-9.

4. Chang GQ, Hao Y, Wong F. Apoptosis: final common pathway of photoreceptor death in rd, rds, and rhodopsin mutant mice. Neuron 1993; 11:595-605.

5. Dryja TP, Rucinski DE, Chen SH, Berson EL. Frequency of mutations in the gene encoding the alpha subunit of rod cGMP-phosphodiesterase in autosomal recessive retinitis pigmentosa. Invest Ophthalmol Vis Sci 1999; 40:1859-65.

6. Berson EL, Rosner B, Sandberg MA, Weigel-DiFranco C, Dryja TP. Ocular findings in patients with autosomal dominant retinitis pigmentosa and rhodopsin, proline-347-leucine. Am J Ophthalmol 1991; 111:614-23.

7. Phelan JK, Bok D. A brief review of retinitis pigmentosa and the identified retinitis pigmentosa genes. Mol Vis 2000; 6:116-24 <>.

8. Rivolta C, Sharon D, DeAngelis MM, Dryja TP. Retinitis pigmentosa and allied diseases: numerous diseases, genes, and inheritance patterns. Hum Mol Genet 2002; 11:1219-27. Erratum in: Hum Mol Genet 2003; 12:583-4.

9. Roof DJ, Adamian M, Hayes A. Rhodopsin accumulation at abnormal sites in retinas of mice with a human P23H rhodopsin transgene. Invest Ophthalmol Vis Sci 1994; 35:4049-62.

10. Bicknell IR, Darrow R, Barsalou L, Fliesler SJ, Organisciak DT. Alterations in retinal rod outer segment fatty acids and light-damage susceptibility in P23H rats. Mol Vis 2002; 8:333-40 <>.

11. LaVail MM, Yasumura D, Matthes MT, Drenser KA, Flannery JG, Lewin AS, Hauswirth WW. Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long-term survival and late-stage therapy. Proc Natl Acad Sci U S A 2000; 97:11488-93.

12. Naash MI, Hollyfield JG, al-Ubaidi MR, Baehr W. Simulation of human autosomal dominant retinitis pigmentosa in transgenic mice expressing a mutated murine opsin gene. Proc Natl Acad Sci U S A 1993; 90:5499-503.

13. Wu TH, Ting TD, Okajima TI, Pepperberg DR, Ho YK, Ripps H, Naash MI. Opsin localization and rhodopsin photochemistry in a transgenic mouse model of retinitis pigmentosa. Neuroscience 1998; 87:709-17.

14. Olsson JE, Gordon JW, Pawlyk BS, Roof D, Hayes A, Molday RS, Mukai S, Cowley GS, Berson EL, Dryja TP. Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa. Neuron 1992; 9:815-30.

15. Huang PC, Gaitan AE, Hao Y, Petters RM, Wong F. Cellular interactions implicated in the mechanism of photoreceptor degeneration in transgenic mice expressing a mutant rhodopsin gene. Proc Natl Acad Sci U S A 1993; 90:8484-8.

16. Humphries MM, Rancourt D, Farrar GJ, Kenna P, Hazel M, Bush RA, Sieving PA, Sheils DM, McNally N, Creighton P, Erven A, Boros A, Gulya K, Capecchi MR, Humphries P. Retinopathy induced in mice by targeted disruption of the rhodopsin gene. Nat Genet 1997; 15:216-9.

17. Kijas JW, Cideciyan AV, Aleman TS, Pianta MJ, Pearce-Kelling SE, Miller BJ, Jacobson SG, Aguirre GD, Acland GM. Naturally occurring rhodopsin mutation in the dog causes retinal dysfunction and degeneration mimicking human dominant retinitis pigmentosa. Proc Natl Acad Sci U S A 2002; 99:6328-33.

18. Mendez A, Burns ME, Roca A, Lem J, Wu LW, Simon MI, Baylor DA, Chen J. Rapid and reproducible deactivation of rhodopsin requires multiple phosphorylation sites. Neuron 2000; 28:153-64.

19. Sung CH, Makino C, Baylor D, Nathans J. A rhodopsin gene mutation responsible for autosomal dominant retinitis pigmentosa results in a protein that is defective in localization to the photoreceptor outer segment. J Neurosci 1994; 14:5818-33.

20. Chen J, Makino CL, Peachey NS, Baylor DA, Simon MI. Mechanisms of rhodopsin inactivation in vivo as revealed by a COOH-terminal truncation mutant. Science 1995; 267:374-7.

21. Cai K, Langen R, Hubbell WL, Khorana HG. Structure and function in rhodopsin: topology of the C-terminal polypeptide chain in relation to the cytoplasmic loops. Proc Natl Acad Sci U S A 1997; 94:14267-72.

22. Deretic D, Schmerl S, Hargrave PA, Arendt A, McDowell JH. Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA. Proc Natl Acad Sci U S A 1998; 95:10620-5.

23. Li T, Snyder WK, Olsson JE, Dryja TP. Transgenic mice carrying the dominant rhodopsin mutation P347S: evidence for defective vectorial transport of rhodopsin to the outer segments. Proc Natl Acad Sci U S A 1996; 93:14176-81.

24. Li ZY, Wong F, Chang JH, Possin DE, Hao Y, Petters RM, Milam AH. Rhodopsin transgenic pigs as a model for human retinitis pigmentosa. Invest Ophthalmol Vis Sci 1998; 39:808-19.

25. Banin E, Cideciyan AV, Aleman TS, Petters RM, Wong F, Milam AH, Jacobson SG. Retinal rod photoreceptor-specific gene mutation perturbs cone pathway development. Neuron 1999; 23:549-57.

26. Petters RM, Alexander CA, Wells KD, Collins EB, Sommer JR, Blanton MR, Rojas G, Hao Y, Flowers WL, Banin E, Cideciyan AV, Jacobson SG, Wong F. Genetically engineered large animal model for studying cone photoreceptor survival and degeneration in retinitis pigmentosa. Nat Biotechnol 1997; 15:965-70.

27. Blackmon SM, Peng YW, Hao Y, Moon SJ, Oliveira LB, Tatebayashi M, Petters RM, Wong F. Early loss of synaptic protein PSD-95 from rod terminals of rhodopsin P347L transgenic porcine retina. Brain Res 2000; 885:53-61.

28. Peng YW, Hao Y, Petters RM, Wong F. Ectopic synaptogenesis in the mammalian retina caused by rod photoreceptor-specific mutations. Nat Neurosci 2000; 3:1121-7.

29. Tso MO, Li WW, Zhang C, Lam TT, Hao Y, Petters RM, Wong F. A pathologic study of degeneration of the rod and cone populations of the rhodopsin Pro347Leu transgenic pigs. Trans Am Ophthalmol Soc 1997; 95:467-83.

30. Kraft TW, Schnapf JL. Aberrant photon responses in rods of the macaque monkey. Vis Neurosci 1998; 15:153-9.

31. Kraft TW, Schneeweis DM, Schnapf JL. Visual transduction in human rod photoreceptors. J Physiol 1993; 464:747-65.

32. Baylor DA, Lamb TD, Yau KW. The membrane current of single rod outer segments. J Physiol 1979; 288:589-611.

33. Lem J, Krasnoperova NV, Calvert PD, Kosaras B, Cameron DA, Nicolo M, Makino CL, Sidman RL. Morphological, physiological, and biochemical changes in rhodopsin knockout mice. Proc Natl Acad Sci U S A 1999; 96:736-41.

34. Baylor DA, Nunn BJ, Schnapf JL. The photocurrent, noise and spectral sensitivity of rods of the monkey Macaca fascicularis. J Physiol 1984; 357:575-607.

35. Lamb TD, McNaughton PA, Yau KW. Spatial spread of activation and background desensitization in toad rod outer segments. J Physiol 1981; 319:463-96.

36. Lamb TD, Pugh EN Jr. A quantitative account of the activation steps involved in phototransduction in amphibian photoreceptors. J Physiol 1992; 449:719-58.

37. Baylor DA, Lamb TD, Yau KW. Responses of retinal rods to single photons. J Physiol 1979; 288:613-34.

38. Xu J, Dodd RL, Makino CL, Simon MI, Baylor DA, Chen J. Prolonged photoresponses in transgenic mouse rods lacking arrestin. Nature 1997; 389:505-9.

39. Cideciyan AV, Zhao X, Nielsen L, Khani SC, Jacobson SG, Palczewski K. Null mutation in the rhodopsin kinase gene slows recovery kinetics of rod and cone phototransduction in man. Proc Natl Acad Sci U S A 1998; 95:328-33.

40. Chen CK, Burns ME, He W, Wensel TG, Baylor DA, Simon MI. Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1. Nature 2000; 403:557-60.

41. Chen CK, Burns ME, Spencer M, Niemi GA, Chen J, Hurley JB, Baylor DA, Simon MI. Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase. Proc Natl Acad Sci U S A 1999; 96:3718-22.

42. Makino CL, et al. Effects of photoresponse prolongation on retinal rods of transgenic mice. In: Williams TP, Thistle AB, editors. Photostasis and related phenomena. Proceedings of Neuroscience Program Symposium on Photostasis and Related Topics; 1997 Feb 21-23; Tallahassee (FL). New York: Plenum Press; 1998. p. 129-51.

43. Tsang SH, Burns ME, Calvert PD, Gouras P, Baylor DA, Goff SP, Arshavsky VY. Role for the target enzyme in deactivation of photoreceptor G protein in vivo. Science 1998; 282:117-21.

44. Burns ME, Mendez A, Chen J, Baylor DA. Dynamics of cyclic GMP synthesis in retinal rods. Neuron 2002; 36:81-91.

45. Calvert PD, Govardovskii VI, Krasnoperova N, Anderson RE, Lem J, Makino CL. Membrane protein diffusion sets the speed of rod phototransduction. Nature 2001; 411:90-4.

46. Green ES, Menz MD, LaVail MM, Flannery JG. Characterization of rhodopsin mis-sorting and constitutive activation in a transgenic rat model of retinitis pigmentosa. Invest Ophthalmol Vis Sci 2000; 41:1546-53.

47. Tamura T, Nakatani K, Yau KW. Calcium feedback and sensitivity regulation in primate rods. J Gen Physiol 1991; 98:95-130.

48. Nakatani K, Tamura T, Yau KW. Light adaptation in retinal rods of the rabbit and two other nonprimate mammals. J Gen Physiol 1991; 97:413-35.

49. Tamura T, Nakatani K, Yau KW. Light adaptation in cat retinal rods. Science 1989; 245:755-8.

50. Schneeweis DM, Schnapf JL. The photovoltage of macaque cone photoreceptors: adaptation, noise, and kinetics. J Neurosci 1999; 19:1203-16.

51. Li ZY, Kljavin IJ, Milam AH. Rod photoreceptor neurite sprouting in retinitis pigmentosa. J Neurosci 1995; 15:5429-38.

52. Moritz OL, Tam BM, Papermaster DS, Nakayama T. A functional rhodopsin-green fluorescent protein fusion protein localizes correctly in transgenic Xenopus laevis retinal rods and is expressed in a time-dependent pattern. J Biol Chem 2001; 276:28242-51.

53. Jin S, McKee TD, Oprian DD. An improved rhodopsin/EGFP fusion protein for use in the generation of transgenic Xenopus laevis. FEBS Lett 2003; 542:142-6.

54. Ohguro H, Rudnicka-Nawrot M, Buczylko J, Zhao X, Taylor JA, Walsh KA, Palczewski K. Structural and enzymatic aspects of rhodopsin phosphorylation. J Biol Chem 1996; 271:5215-24.

55. Ohguro H. High levels of rhodopsin phosphorylation in missense mutations of C-terminal region of rhodopsin. FEBS Lett 1997; 413:433-5.

56. Kisselev OG, Downs MA, McDowell JH, Hargrave PA. Conformational changes in the phosphorylated C-terminal domain of rhodopsin during rhodopsin arrestin interactions. J Biol Chem 2004; 279:51203-7.

57. Liu P, Roush ED, Bruno J, Osawa S, Weiss ER. Direct binding of visual arrestin to a rhodopsin carboxyl terminal synthetic phosphopeptide. Mol Vis 2004; 10:712-9 <>.

58. Zhang L, Sports CD, Osawa S, Weiss ER. Rhodopsin phosphorylation sites and their role in arrestin binding. J Biol Chem 1997; 272:14762-8.

59. Baylor DA, Hodgkin AL, Lamb TD. The electrical response of turtle cones to flashes and steps of light. J Physiol 1974; 242:685-727.


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