Proc. Nat. Acad. Sci. USAVol. 72, No. 1, pp. 381-385, January 1975

Light-Stimulated Phosphorylation of Rhodopsin in the Retina: The Presenceof a Protein Kinase That Is Specific for Photobleached Rhodopsin

(protein phosphorylation/cAMP/cGMP)

MALCOLM WELLER, NOELLE VIRMAUX, AND P. MANDEL

Centre de Neurochimie, 11 Rue Humann, 67085 Strasbourg Cedex, France

Communicated by Seymour S. Kety, September 30, 1974

ABSTRACT A protein kinase has been extiacted frombovine rod outer segments by a mild procedure. The en-zyme acts specifically on photobleached, not unbleached,rhodopsin and will not catalyze the phosphorylation ofhistones, phosvitin, or casein. We propose the name"opsin kinase" for the enzyme, which is not affected bycyclic nucleotides but which is inhibited by theophylline.Preparations of purified rod outer segments, however,appear to contain only low concentration of opsin phos-phatase activity.

It has recently been shown in several laboratories that whenrod outer segments (ROS) prepared from frog (1, 2) or ox(3-5) retinas are incubated with ATP and Mg2+ rhodopsin isphosphorylated and the reaction is markedly stimulated bylight.There seem to be three ways in which light could act. First,

by directly stimulating the activity of the kinase, second, byaltering the conformation of the rhodopsin so that it becomes asubstrate for the kinase, and third, by altering the concentra-tion of some cofactor which changes the activity of the kinase.

In support of the third possibility, it has been observed thatthe rate of production of 3': 5'-cAMP and cGMP in ROS islowered on exposure to light (6-10) and, since rates of proteinphosphorylation are, in so many cases, controlled by cyclicnucleotides, it was at first thought that the effect of light oncyclic nucleotide concentration could be correlated with theeffect on protein phosphorylation (3, 4). A decrease in cyclicnucleotide concentration could increase the net rate of proteinphosphorylation if the cyclic nucleotide inhibited a proteinkinase or, alternatively, stimulated a protein phosphatase.Cyclic-AMP-inhibited protein kinases have been discoveredin the slime mold (11) and cAMP-stimulated protein phos-phatases in the toad bladder cell membrane (12).

In this paper, we show that neither cAMP nor cGMP haveany effect on the phosphorylation of ROS protein. The proteinkinase in ROS is specific to photobleached, not unbleachedrhodopsin, and the increase in substrate concentration whichresults from exposure of ROS to light is alone sufficient toaccount for the increase in protein phosphorylation.

MATERIALS AND METHODS

Preparation of Rod Outer Segments from Bovine Retinas wasas previously described (13). The segments routinely showed aratio of absorbance at 280 to 500 nm of about 2.2. Incubationwith 1 1-cis-retinaldehyde only increased the absorption at 500nm by 5-10%, indicating that the material was only 5-10%bleached.

Abbreviation: ROS, rod outer segments.

Preparation of Tris Extracts from ROS (unless otherwisestated all extractions were performed at 40 in dim red light).ROS were homogenized by hand at a concentration of about0.5 mg of protein per ml in 0.01 M Tris*HCl (pH 7.0) andcentrifuged at 100,000 X g for 60 min.

Preparation of "Purified Rhodopsin" by Differential Extrac-tion ofROS with Sodium Dodecyl Sulfate. The pellet remainingafter extraction with Tris - HCl was extracted twice with 0.1%osodium dodecyl sulfate in 10 mM Tris*HCl (pH 7.0). Theinsoluble material was washed three times with 0.066 M Naphosphate buffer, pH 7.0. This procedure removes not onlynonrhodopsin proteins but also bleached rhodopsin. On thebasis of polyacrylamide gel electrophoresis opsin appears to bethe only protein in the preparation (14).

Protein Determination was by the method of Lowry et al.(15) using bovine plasma albumin as standard.

Radioactive ATP 7y-labeled [32P]ATP was prepared (16) andpurified (17) by previously described methods.

Measurement of Protein Phosphorylation. All incubationswere carried out in the presence of 50 mM Tris * HCl pH 7.4, 1mM [a2PIATP and 1 mM MgCl2 (unless otherwise stated) at370 in a volume of 0.5 ml with 100-200 ltg of protein. Reactionswere terminated by the addition of 2 ml of ice-cold 10%trichloroacetic acid (or 20% to precipitate histones) and 0.1mg (0.1 ml) of bovine serum albumin added as protein carrier.The protein precipitate was washed twice with 10% trichloro-acetic acid, M orthophosphoric acid, then resuspended in 0.5ml of 0.1 N NaOH and incubated for 10 min at 37°, after which2 ml of 10 or 20% trichloroacetic acid was added and the pro-tein was spun down. The protein precipitates were washed oncein 1 ml of ethanol-ether (1/1) and dissolved in 2 ml of 0.1 NNaOH at 1000 and the Cerenkov radiation was measured (18).

Measurement of Protein Dephosphorylation. Samples of ROSwere incubated with 1 mM MgCl2, 1 mM [a2P]ATP and 50mM Tris HCl (pH 7.4) at a protein concentration of about0.4 mg/ml for 30 min at 37°. The suspension was then centri-fuged at 100,000 X g for 10 min and the pellet was washedtwice by resuspension and centrifugation from 50mM sodiumphosphate buffer, pH 7.0, before final resuspension at a con-centration of 1-2 mg of protein per ml in the same buffer.Aliquots of the [82P]ROS were then incubated with 50 mMTris-HCl (pH 7.4) at a protein concentration of about 0.4mg/ml with the additions stated in the text. Aliquots (0.5 ml)were then taken at different times and mixed with 2 ml of ice-cold 10% trichloroacetic acid, the denatured protein beingwashed and its radioactivity measured as described above.

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Proc. Nat. Acad. Sci. USA 72 (1975)

o a0% bleach0~-0

0

rC12L I

L 4 /%50/ bleach

m It

o A~~~~24%blech

Va-0

I0

L

0

E

c0

mIt _ ----4 _ I .._.__C

X A DarkA

° 10 30 50 70 90 1106 12 18 24 30 c Relative percentage oFTime (min) unbleached rhodopsin

FIG. 1. The effect of bleaching on the posphorylation ofrhodopsin. (a) Samples of purified rhodopsin were suspended atabout 1 mg of protein per ml in 50 mM sodium phosphate buffer(pH 7.0) and bleached to various extents by exposure to whitelight. Aliquots were taken to determine the extent of bleaching bymeasuring the decrease in absorbance at 500 nm and the remain-ing material was incubated under the phosphorylation conditionsdescribed in the text. Broken lines show the time course of phos-phorylation of intact ROS, A in dim red light; A or in white light.Similar results were obtained with three separate experiments.(b) The maximum amount of phosphate which could be in-corporated into rhodopsin which had been bleached to variousextents was plotted against the amount of unbleached rhodopsinin the sample, shown as a percentage of the amount in thematerial before exposure to light (relative percentage of un-

bleached rhodopsin). Results are shown as means standarddeviations.

RESULTSIn agreement with the results of other workers (1-5), we foundthat isolated ROS-on incubation with [32P]ATP show a light-stimulated incorporation of 32p (Fig. 1). The effect of light ismuch more pronounced than that observed by Frank et al. (4)who, after 2-min incubation, only obtained a stimulation ofabout 1.3 times while we obtained a stimulation of 10-20times, in agreement with other workers (1, 3, 5).The incorporated radioactivity is bound to protein and not

lipid, since it could neither be extracted into chloroform-methanol (2/1) nor into chloroform-methanol-concentratedHCl (300/200/1). Polyacrylamide gel electrophoresis in thepresence of sodium dodecyl sulfate showed, in agreement withother workers (1-5), that opsin was the major, if not the only,protein to be phosphorylated. It has been found that serineand to a lesser extent threonine are the major, if not only, siteof phosphorylation (3, 5).Other workers have reported, without giving any details,

that the phosphorylation of ROS is unaffected by cAMP(3, 4). The effect of cGMP, which is also present in high con-

centrations in ROS (8), was not examined. To examine theeffects of cyclic nucleotides on the phosphorylation of ROS itwas necessary to add a phosphodiesterase inhibitor to preventthe hydrolysis of the cyclic nucleotide.We found that theophylline and SQ 20009 (which is struc-

turally very similar to theophylline) are both quite stronginhibitors of ROS phosphorylation in the light but have lesseffect in the dark, while papaverine is only slightly inhibitoryin the light and has no effect in the dark (Table 1). NeithercAMP nor cGMP have any effect in the presence or absence ofphosphodiesterase inhibitors in the light or in the dark, andsimilarly dibutyryl cGMP has no effect in the light or dark,while dibutyryl cA;M.P, though having no effect in the dark,

TABLE 1. The effect of phosphodiesterase inhibitors and cyclicnucleotides on the intrinsic protein kinase activity of ROS

Phosphodiesterase Cyclic % of controlinhibitor nucleotide (no additions)

White lightSQ 20009 - 61 i 2.4 (6)SQ20009 1 mM cAMP (I mM) 58.5 ±4(6)SQ 20009 cGMP (0. 1 mM) 55.5 i 3.8 (4)Theophylline - 61. 5 ± 7.1 (6)Theophylline 10 mM cAMP (1 mM) 61.8 ± 6 (4)Theophylline cGMP (0.1 mM) 60, 70Papaverine - 89 ± 8.2 (7)Papaverine 2 mM cAMP (1 mM) 79 i 12 (4)Papaverine cGMP (0. 1 mM) 85, 91

cAMP (I mM) 94.8 ±i 8 (6)- cGMP (0. I mM) 93 i 4.6 (3)- Dibutyryl cAMP 77.2 i 11.6 (10)

Dibutyryl cGMP 102. 5 i 4. 1 (4)Dim red light

SQ 20009 95 ±t 5 (4)SQ 20009 1 mM cAMP (I mM) 91.2 i 4.7 (6)SQ 20009 cGMP (0. I mM) 102Theophylline 86. 7 ± 13.1 (6)Theophylline 10 mM cAMP (1 mM) 99. 3 i 1. 2 (3)Theophylline cGMP (0.1 mM)Papaverine - 106.25 ±- 4.8 (4)Papaverine 2 mM cAMP (1 mM) 114.3 t 27 (4)Papaverine cGMP (0. 1 -mM)

- cAMP (I mM) 104 ± 9 (5)cGMP (0. 1 mM) 97.7 ± 7.9 (4)Dibutyryl cAMP 98.9 ± 11.6 (10)Dibutyryl cGMP 99.3 ± 15.5 (4)

Protein phosphorylation was carried out as described in thetext for 2 min either in white or dim red light. Results are shown asmeans ± standard deviations with the number of observationsshown in parentheses.

was slightly inhibitory in the light (Table 1). The effect ofphosphodiesterase inhibitors could be due to an increasedaccumulation of cyclic nucleotides, which cause an inhibitionof phosphorylation. Cyclic nucleotides, however, do not affectphosphorylation, but it may be that it is difficult for them toreach the kinase inside the ROS membranes. To solve thisproblem we examined the effect of cyclic nucleotides on thesoluble kinase (see below).

Extraction of Protein Kinase Activity from ROS. It haspreviously been reported that rhodopsin kinase activity maybe extracted from ROS by sonication in Tris EDTA (5). Wetreated ROS by a milder procedure, described in the Materialsand Methods section. About 8-10% of the total ROS proteinis solubilized and the insoluble material remaining after ex-traction showed a loss of intrinsic protein kinase activity,particularly when measured in the light. The activity wasrestored by addition of the Tris extract (Table 2) which didnot, however, show any self-phosphorylation.

It was also found that the Tris extract catalyzed the phos-phorylation of purified photobleached, but not unbleached,rhodopsin (Fig. 1). Exposure of the Tris extract to light didnot cause it to phosphorylate unbleached rhodopsin, nor did itaffect the rate with which it could catalyze the phosphoryla-

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An Opsin-Specific Protein Kinase in Retinal Rods 383

tion of bleached rhodopsin. The Tris extract thus contains aprotein kinase which is itself unaffected by light but whichcatalyzes tbe phosphorylation of bleached rhodopsin.

If ROS were illuminated for 2 min before Tris treatment thequantity of protein that could be extracted was reduced fromabout 60 to 20 to 30 mg/g of total rhodopsin protein. Moreinterestingly, the extract prepared in this way contained muchless protein kinase activity (Fig. 2).

This could be explained if the kinase binds rather stronglyto its substrate, bleached rhodopsin. Since there is much more

of this substrate in light-exposed ROS, it is easier to extractthe enzyme from material kept in the dark.

Because of the higher concentration of protein kinase activ-ity all the Tris extracts referred to below as soluble enzyme

were prepared from material kept in the dark.

The Effect of Cyclic Nucleotides on Protein Kinase Activity ofTris Extract Prepared from ROS. Working with a mixture ofsoluble enzyme and purified rhodopsin, it was possible toexamine the direct effect of cyclic nucleotides on the proteinkinase. As was the case with intact ROS, it was found thattheophylline and SQ 20009 were strongly inhibitory to thephosphorylation of the purified rhodopsin, while cAMP or

cGMP alone, or in the presence of phosphodiesterase inhibi-tors, have no effect (Table 3). It thus appears that the effect oftheophylline (or the derivative SQ 20009) on the phosphoryla-tion of intact ROS was not due to an increase in cyclic nucle-otide concentration caused by the inhibition of phosphodies-terase activity, but was due to a direct inhibition of proteinkinase activity. The fact that more inhibition is observed inthe light than in the dark can be explained by the fact thatbleached rhodopsin is the substrate for the enzyme. In thedark the concentration of bleached rhodopsin is very low so

that the protein kinase is present in excess; partial inhibitionof its activity would not cause a great reduction in proteinphosphorylation. In the light, however, there is much more

bleached rhodopsin substrate so that the enzyme is no longerpresent in excess, hence inhibitors would cause more reductionof protein phosphorylation.

Specificity of the Protein Kinase Activity of Tris ExtractsPreparedfrom ROS. There have been reports of the occurrence

in whole retina and even in isolated ROS of an enzyme that isstimulated by cAMP and that catalyzes the phosphorylationof histones (5, 19, 20, 22). The lack of effect of cAMP on the

TABLE 2. Restoration of intrinsic protein kinase activity toTris-extracted ROS

Intrinsic protein kinase activity*

Material Dim red light White light

Dark-extracted ROS 75 ±4 20 5Dark extract 1 3 2 5Dark-extracted ROS plusdark extract 73 5 74 4

ROS were extracted with 10 mM Tris *HCl and proteinphosphorylation was carried out for 2 min as described in the text.Where Tris extract was added it was at a concentration of 30,ugof protein per ml. Results are shown as mean ±- standard devi-ation and are taken from four experiments.

* % of activity of untreated ROS measured in dim red lightand white light.

-E c- 8 /

L 4;i40 a.

Coo

0

`0 oE0 20 40 60 80

C pg of tris extract protein

FIG. 2. Opsin kinase activity of Tris extracts from ROS.ROS were extracted with 10 mM Tris-HCl as described in thetext either in the dark (0), or after the ROS were exposed for 2min to white light (0). The protein kinase activity of the extracttowards purified bleached rhodopsin was then determined asdescribed in the text, using 100 ,ug of rhodopsin and a reactiontime of 4 min. Similar results were obtained with. three separateexperiments.

phosphorylation of rhodopsin catalyzed by the Tris extractindicates that the protein kinase responsible for this reaction isprobably not the same as that which catalyzes the phospho-rylation of histones. In addition it was found that the Trisextract showed slight if any activity towards histones, casein,or phosvitin, even in the presence of cyclic nucleotide (Table4). Similarly we found that a histone kinase purified from beefkidney (21) could not catalyze the phosphorylation ofbleached or unbleached rhodopsin either in the presence orabsence of cyclic AMP. A similar result has recently beenreported for a beef muscle histone kinase (20).The time course of protein phosphorylation of intact ROS is

essentially the same as the time course of the phosphorylationof purified bleached rhodopsin catalyzed by a Tris extractwhen the two are recombined in the same proportion as in theintact material, (Fig. la). There is thus no evidence that opsin

TABLE 3. The effect of phosphodiesterase inhibitors and cyclicnucleotides on the opsin kinase activity of Tris extracts prepared

from ROS

Cyclic % of activityPhosphodiesterase nucleotide in control

inhibitor (0. 1 mM) (-additions)

White lightSQ 20009 1 mM 21, 20SQ 20009 1 mM cAMP 18, 17

cAMP 100, 103SQ 20009 1 mM cGMP 17, 18.5Theophylline 10mM - 51.5 4 6.3 (6)Theophylline 10 mM cAMP 50.7 4 4.1 (6)

- Dibutyryl cGMP 100,97Dibutyiyl cAMP 98 ± 2 (4)

Papaverine 2 mM - 88.25 4± 6.4 (4)Papaverine 2 mM cAMP 75.5 ± 7.5 (4)

Dim red lightSQ 20009 1 mM - 45,41SQ 20009 1 mM cAMP 60,45

- cAMP -98, 94

Incubations were carried out for 2 min as described in the textwith 200,g/ml of rhodopsin and 32 ,ug/ml of Tris extract protein.Results are shown as means 4 standard deviations with thenumber of observations in parentheses.

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TABLE 4. Protein kinase activity of Tris extracts from ROS towards various protein substrates

Protein kinase activity, nmol/min per mg Tris extract protein

8 mM MgC12 1 mM MgC12

+ 10pM cAMP -cAMP + 10,McAMP -cAMP

Histone 0.08 A 0.08 (8) 0.05 4 0.06 (6) 0.8 i 0.8 (6) 0.5 0.6 (4)Phosvitin 0.0440.06(6) 0.09 4 0.1Casein 0.1 4 0.1Bleached rhodopsin 16 4 (6) 17 4 5 (6)

Protein kinase activity was determined as described in the text with 50-100 ,sg of Tris extract protein per ml. Results are shown asmeans A standard deviations with the number of observations in parentheses.

is phosphorylated in the intact ROS by enzymes apart fromthe "opsin kinase" found in the Tris extract.

Effect ofRhodopsin Photostimulation on Protein Phosphoryla-tion. If "opsin kinase" acts only on bleached rhodopsin,exposure of ROS to light will increase the amount of substrateavailable to the enzyme and hence the net amount of phos-phate which can be incorporated into the ROS protein willalso be increased. To demonstrate this point purified rhodopsinwas exposed to light for various times, producing variousdegrees of bleaching, and the time course of phosphorylationwas determined. It was found that increased rhodopsin bleach-ing increased the amount of phosphate which could be in-corporated (Fig. 1). Both the initial rate of phosphorylationand the total amount of phosphate which could be incorpo-rated were increased. No alteration in the time course of phos-phorylation was observed if the bleached rhodopsin was leftin the dark for times up to 10 min before the start of phos-phorylation. This result indicates that the increased phos-phorylation is due to an increased concentration of substratefor the kinase reaction. To make this point clear the amount ofphosphate which could be maximally incorporated into rho-dopsin was plotted against the percentage of unbleached rho-dopsin in the sample. A completely linear relationship was

observed (Fig. lb), showing that the amount of phosphatewhich can maximally be incorporated into rhodopsin isdirectly proportional to the number of bleached rhodopsinmolecules present. This result is in agreement with that of

*' 181

CL0-I0

E3 16E

x

E 140LU

~~a0

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_

7 14 21Time (min)

_1000

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N 80

0

X( 40

o 0o 10 20Time (min)

FIG. 3. Dephosphorylation of ROS proteins. (a) 32P-labeledbleached ROS were incubated in dim red light at 370 at a proteinconcentration of 300 ,ug/ml with 50 mM Tris. HOl (@), with

2-mM MgCl2 (0), or with 1 mM EDTA (A). (b) 3P-labeled ROSwere added at a concentration of 0.4 mg of protein per ml to 50

mM Tris.HCl pH 7.4, 2 mM MgC12 in the presence (/v) or

absence (0) of a homogenate of whole retina prepared at a

concentration of 0.5 retinas per ml of reaction mixture. Similar

results were obtained with three separate experiments.

Frank et al. (4), who showed that there was a roughly linearrelationship between the rate of phosphorylation of ROS andthe extent of bleaching, but is in contrast to the results ofBownds et al. (1), who initially reported that ROS preparedfrom frog retinas showed maximal phosphorylation after only1% of the rhodopsin is bleached. In a recent publication,however, they reported that further bleaching did cause anincrease in phosphorylation, though small bleaches were muchmore effective (2, 22). Our experiments give no indication ofsuch an effect. The method which we used for the preparationof rhodopsin extracts bleached material (14). Examination ofFig. lb shows the line to cross the abscissa at 101%, suggestingthat the preparation of rhodopsin is only 1% bleached beforeexposure to light. Intacts ROS show more phosphorylation inthe dark than does a mixture of rhodopsin and Tris extract(Fig. la).

Several workers have observed a maximal incorporation ofabout 1 mole of phosphate/mole of rhodopsin (1, 3, 5). We,however, like Franks et al. (4), found a maximal incorporationof only about 0.5 mole/mole of bleached rhodopsin. It istempting to explain this result by suggesting that the rhodop-sin used in our experiments was already partially phosphoryl-ated.

In contrast to these observations Bownds et al., workingwith ROS from frog retinas, have reported that at bleaches ofless than 5%, 10-15 moles of phosphate may be incorporatedper mole of bleached rhodopsin. With higher amounts ofbleaching, only 1 mole of phosphate can be incorporated, andthey suggest that at low levels of bleaching phosphate may bebound to unbleached rhodopsin molecules (2). Our experi-ments with cattle retinas give no indication of such an effect.

Dephosphorylation of Rhodopsin. If rhodopsin phosphoryla-tion has any physiological importance, enzymes must bepresent which can not only catalyze the phosphorylation ofrhodopsin but also the dephosphorylation, otherwise, in vivo,the turnover of rhodopsin phosphate could not occur.

It may be seen from Fig. 3a that ROS contain only slightprotein phosphatase activity, which is increased in the pres-ence of Mg2 + and inhibited by EDTA. cAMP had no effect onthe dephosphorylation and the same rate of reaction was ob-served whether the experiment was carried out in the light or

in the dark. Other workers similarly have shown only very lowrates of dephosphorylation (4, 5).

Regeneration of phosphorylated bleached ROS did notalter their rate of dephosphorylation.The low rate of dephosphorylation of phosphorylated rho-

dopsin is a matter for some surprise. Perhaps the protein

'qQAUV= Biochemistry: Weller et al.

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An Opsin-Specific Protein Kinase in Retinal Rods 385

phosphatase activity is soluble and lost during the preparationof the ROS. If phosphorylated ROS were incubated at 370with a crude retinal homogenate, they were certainly dephos-phorylated more rapidly (Fig. 3b) but the reaction was stillnot very fast.

DISCUSSION

The results described in this paper show that ROS contain aprotein kinase which is readily solubilized and which specifi-cally catalyzes the phosphorylation of photobleached but notunbleached rhodopsin. Most protein kinases so far reportedfall into two broad groups: those that catalyze the phos-phorylation of phosvitin, and those that catalyze the phos-phorylation of histones, though, of course, both groups ofenzymes catalyze the phosphorylation of a number of otherproteins as well. The protein kinase present in the Trisextract prepared from ROS belongs to neither group, since itcan catalyze the phosphorylation of neither histones norphosvitin. It appears that it is an enzyme that specificallycatalyzes the phosphorylation of bleached rhodopsin, and wepropose the name "opsin kinase." The fact that the proteinkinase acts only on bleached rhodopsin explains the observa-tion that exposure to light increases the phosphorylation ofROS protein.The fact that ROS proteins are phosphorylated through the

action of a specific protein kinase is one of considerable inter-est. The synaptic membrane proteins of brain are phos-phorylated through the action of a tightly bound proteinkinase which differs in properties in several respects from theenzyme that catalyzes the phosphorylation of phosvitin orhistones (23-25). Like opsin kinase it is inhibited by theophyl-line but not by papaverine (23, 25, 26). It is, however, stimu-lated by cAMP (26, 27).The time course of rhodopsin phosphorylation is much too

slow to be involved in the direct response to light but it may bepostulated that the phosphorylation is concerned in the adap-tation of the photoreceptors to light or dark conditions (5, 22).It has been suggested before (25, 27-29) that the phosphoryla-tion of certain membrane proteins may control passive perme-ability to certain ions and it may be that the phosphorylationof rhodopsin may similarly control passive permeability inROS, so mediating the responsiveness to a light impulse.We thank G. Nullans for excellent technical assistance and

Dr. S. M. Hess (Squibb Institute of Medical Research) for thegift of the compound SQ 20009. M.W. gratefully acknowledgesreceipt of a Science Research Council Fellowship.

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