Rhodopsin and the visual process

Biochimica et Biophysica .4cta, 463 (1977) 91-125 © Elsevier/North-Holland Biomedical Press B B A 86039

RHODOPSIN SANFORD

AND

THE

VISUAL

PROCESS

E. O S T R O Y

Department o f Biological Sciences, Purdue University, West LaJayette, Ind. 47907 (U.S.A.) (Received July 26th, 1976)

CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

91

II.

R h o d o p s i n : intermediates a n d c o n f o r m a t i o n changes . . . . . . . . . . . . . . .

93

A. Intermediates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

93

B. T h e sequence o f intermediates in vertebrate r h o d o p s i n . . . . . . . . . . . . .

96

C. C o n f o r m a t i o n changes of r h o d o p s i n during photolysis . . 1. General changes . . . . . . . . . . . . . . . . . . . 2. C o m p a r i s o n s of digitonin extracts a n d intact systems . 3. T h e p h o s p h o r y l a t i o n of vertebrate r h o d o p s i n . . . . .

97 97 102 104

. . . .

. . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

D. R h o d o p s i n as a m e m b r a n e protein . . . . . . . . . . . . . . . . . . . . . . I. Location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. M o v e m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

105 105 196

II1.

R h o d o p s i n c h a n g e s a n d retina electrical signals . . . . . . . . . . . . . . . . . .

107

IV.

H y d r o g e n ion effects of the photoreceptor cell . . . . . . . . . . . . . . . . . .

109

V.

C a l c i u m effects of the photoreceptor cell . . . . . . . . . . . . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111 111

B. Vertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I 11

C. Invertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

112

VI.

Cyclic nucleotide effects of the photoreceptor cell . . . . . . . . . . . . . . . . .

115

VII.

T h e purple m e m b r a n e of the Halobacterium halobium . . . . . . . . . . . . . . .

117

VIII.

Hypothesis of visual photoreceptor function . . . . . . . . . . . . . . . . . . .

119

Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

119

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

120

I. I N T R O D U C T I O N The visual system serves as the focal point for a wide variety of interests. is a n i m p o r t a n t

sensory process which tranduces

Abbreviations used as follows: acid; Meta, m e t a r h o d o p s i n .

EGTA,

Vision

light energy into electrical energy.

ethyleneglycolbis(~-aminoethyl ether)-N,N'-tetraacetic

92 The vertebrate retina develops from brain tissue and the receptor molecule of the visual system is a membrane bound chromophoric-lipo-glycoprotein. Thus whether one is interested in energy transduction, the function or interaction of brain cells, or the function of membrane bound proteins, the visual system can be a representative example. Moreover, for these studies the visual system has some distinct advantages. The cells of the retina can be studied individually and the light stimulus can be regulated for energy, intensity and pattern. Also the visual pigments are clearly identified by their ability to absorb the stimulus photons and some of their conformations can be observed by examining the spectral changes induced by illumination. One of the crucial questions in the vision field has been the mechanism of action of the photoreceptor cell. Only a few of the critical steps in this precess are understood. Light is absorbed by the visual pigment and subsequently the sedium conductance and then the potassium conductance of the photoreceptor are altered. Upon illumination of the vertebrate photoreceptor the sodium conductance of the plasma membrane of the outer segment is reduced [l-3] and the hyperpolarization caused by this conductance change causes an increase in conductance in the inner segment to an ion with an equilibrium potential around - - 8 0 mv [4], most likely potassium. The resting potential of the cell is approximately - - 3 0 mv and with high intensity illumination (approx. 100 quanta per rod) the potential may go transiently to - - 5 0 mv and level at about - - 4 0 mv [5]. The half-maximum amplitude occurs typically at a light intensity of only 30 photons per rod [6]. In the invertebrate the sodium conductance [7] and the calcium concentration of the cell increase on illumination [8]. The internal calcium concentration appears to control the sensitivity (the amplitude of the receptor response per unit of simulus energy) of the cell by regulating its sodium conductance [9-1 I]. The resting potential of the Limulus ventral photoreceptor is normally about --48 mv and on illumination the potential rises transiently to - - 8 mv and levels at --28 mv [12]. The Drosophila photoreceptor appears to have somewhat different permeabilities or ion concentrations since its resting potential is approximately - - 2 0 to 40 mv and with high intensity light the membrane potential goes transiently to 0 mv and levels at - - 1 0 mv [13]. There is a substantial amount of additional data about the visual pigments and various aspects of the photoreceptor cell, but except for maintenance processes (such as the sodium-potassium pump, or visual pigment or disk renewal) the importance of this data to the functioning of the photoreceptor cell is not yet clear. The purpose of this review is to summarize and correlate the data which may relate to the functioning of the photoreceptor. There is an emphasis on the role of the visual pigment, and the maintenance processes are not discussed in any detail. I have tried to keep the interpretation of the data to a minimum so that each person can incorporate the data they wish into their own view of visual function. Other recent reviews which correlate biochemical and electrophysiological data are Goldsmith [14], Shichi [15], Ebrey and Honig [16] and Stieve [17].

93 11. R H O D O P S I N :

INTERMEDIATES

AND

CONFORMATION

CHANGES

IIA. Intermediates The initial step of the visual process is the absorption of light by the visual pigment. In all cases so far investigated the native visual pigment consists of an 11-cis retinal (vitamin A~ or A2 aldehyde) bound to a protein called opsin. This class of molecules, regardless of origin, have generally been called rhodopsin (for vitamin A~ compounds) or porphyropsin (for vitamin A2 compounds) following the terminology of Wald [18, 19]. Considering the similarity of their chromophore, the range of absorption peaks exhibited by the various visual pigments and their photoproducts is surprisingly diverse. A partial list of some of the rhodopsins, their spectral changes and rates of reaction on illumination is presented in Figs. 1 and 2. The vertebrate rod visual pigment, rhodopsing9s, exhibits the most complex sequence of spectral intermediates on illumination (Fig. 1). In contrast, the vertebrate principal cone visual pigment, a rhodopsins75, exhibits no photoproducts in the 380-725 nm region on illumination (Fig. 2). The invertebrate visual pigments exhibit one photoproduct (as in the pigment of Drosophila (ref. 39, Fig. 2), blowfly [41] or octopus [41]), or a limited number of photoproducts (as in Limulus or owlfly, Fig. 2) though there have not been extensive low temperature studies in these systems. Since the spectral properties of the rhodopsin are based on light absorption by the retinal chromophore, the native rhodopsin spectra and the thermal intermediates must represent altered interactions between the retinal and the protein and lipid of the opsin (including the Schiff base binding site (-C--N-)). The effect on the spectrum of protonation or deprotonation of the Schiff base retinal-protein bond is well documented. Simple Schiff bases between retinal and methylamine have absorption peaks at 365 nm for the deprotonated form and at 440 nm for the protonated form [50]. In the vertebrate rhodopsin as well as the invertebrate Limulus (ref. 42, Fig. 2), owlfly (ref. 41, Fig. 2), and squid [51] rhodopsin, there are acid-base sensitive intermediates based on the state of protonation of the Schiff base. Schiff base deprotonation seems responsible for the Lhnulus, owlfly, and squid, Acid Meta478-50o to Alkaline Meta38o transition (Fig. 2, ref. 51). In the vertebrate, N-retinylidine opH~

sin44o ~- N-retinylidine opsin365 +H + is observed at the extremes of pH, and appears to be a simple Schiff base complex (Fig. 2, refs. 21, 50 and 52). Also ~n the vertebrate, the spectral changes associated with the Meta 1478 tO Meta II3so reaction, as well as the Meta II38o to Meta Ili405 reaction, appear to be associated mainly with the deprotonation and then protonation of the Schiff base bond [22, 52, 53]. The other retinal-protein interactions that are responsible for the spectral deviations from the simple Schiff bases are not clear. Protonation and deprotonation of other ionizable groups accompany (and in some cases affect) some of the rhodopsin intermediate reactions (Section lI C-l, refs. 20-22 and 52). Raman spectroscopic and model studies suggest an interaction of some of the aromatic groups in the protein

t~

¢3 O ~'-- O

S~

©E

~g

_g

E~

~g

P e~ e

Hydrolysis Pathway Maximum Percentages 30~ Digitonin(3°~ 27Z Frog retina(25 ) 20% Frog r e t i n a ( 1 0 ° ) 251 Human retina

h~vh'o k>540

11-cls or ll-els, 12-S-cis retinal M.W. 35-40,000 pH 5.4-7.7

I478

Retlnol

$

# $

----~.Retinal •

Opstn

H2 o

NRO440 pH_ >7.7. NR0365 pH<5.5

:>.+15oc

Metarhodopsln III465 (Para)

>-5°C

----- Metarhodopsin I1380

Metarhodopsin

IIl>0°c

Lumirhodopsin497

Bathorhodopsin543 q ~ ~'~" Hyps orhodopsin430 (Pre lumirhodops In)

hv

Rhodopsin498

Vertebrate Rhodopsin (Refs. 20-29)

107,600(13 ° )

4076(20 °)

578 & 2772 x 10 -6 (37 ° )

3 1 . 5 & 256 & 3590 x 10 - 6 (37 ° )

50 x 1 0 - 9 ( 2 5 ° , T r i t o n )

30 x IO'9(25°,DDAO)

Bovine in Digitonin (Refs. 21,22,30-33 )

(sec.)

~In0(37°,Rat)

63(37°,Rat)

13,860 & 5Z,750 x I0 -v (20O,Bovine ROS~

250 x lO-6(37°,Rat)

16 x I0 "6 (18°,Bovine ROS)

13 & 78 & 280 x 10 -9 (38°,Bovine ROS)

ROS/Retina (Refs. 23,31,34)

Nalf-lives

Human Retina (Ref. 29 )

(23 ° )

267(21 °)

495(21 °)

60.8

23.1(36 ° )

173(36 ° )

$7. : (21 c, 77 (36 ° ) direct to retinal) 11.8 (23 ° possibly to retinal)

49.~ _1

550 x I0-6(23 °)

50 x 10 -6 (20 °, Frog)

170 x I0 "9 (20 ° , Frog)

Fro$ Retina (Ref~, 34,3537)

e~

g

B

5'

o o

c

hv

X463~Actd

I

"

t½ - i00 msec

Metarhodopsin580

t½ - I0 msec

hv

Alkal~ne Metarhodopsin380

pH>9.61[pH 7.8

Hetarhodopsins00

I

Prelumi

l

Rhodopsin520_530

hv

Rhodopsin480 (M.W. 37,004

/

H+

+tt+

Alkaline Hetarhodopsin380

>-90Oc

L550

[ >-130°c

(N520)~-M412 (unprotonated Schlff base) >-50oC

0640

K590

B~cteri°rh°d°psin570 (all-trans) II 560 (13-cis protonated Schiff base M.W. 26,000)

Bacteriorhodopsin ~Halobacterium halobium) (Scheme is for llght adapted (all-trans) cycle) (Refs. 44-49).

--Acld Metarhodopsln460 (ll-cis)

495

Metarhodopsin

pK 9.2

Acid Metarhodopsin478 ~

Lumirhodopsin375

II 0o

~Rhodopsin345 (M.W. 35,000)

Owlfly (Ascalaphus marcaronlus) (Ref, 41)

Barnacle (Balanus (Ref. 43; T = 5-(])

Drosophila (Drosophila melano~aster) (Refs. 39, 40)

Limulus (Limulus polyphemus) (Ref. 42; T - 25°C)

No photoproducts detected 380-725 run (Principal cones) Some accessory cones gave 385-390 nm peaks

I h'J

Rhodopsin575

(Ref. 38)

Frog cone (R. pipiens)

96 with the retinal [53-55]. Molecular orbital calculations, based on an assumed interaction of a charge on the protein with the chromophore, have given results which are consistent with some of the observed spectral shifts [56, 57]. Even though the spectral intermediates represent only the retinal-protein interactions, it seems that these interactions reflect the protein conformation changes that occur in rhodopsin. There is no clear evidence for rhodopsin conformation changes occurring on a time scale different from the spectral intermediate changes. Also since the thermal intermediate sequence is qualitatively and kinetically reproducible under controlled conditions, the intermediates are a detailed time record of the lightinduced conformation changes of rhodopsin. For such detailed conformation data, the rhodopsins are even more advantageous than hemoglobin, since in the rhodopsin there are more spectrally distinct states and they are more easily isolated and studied. liB. The sequence of intermediates in vertebrate rhodopsin The sequence of intermediates for vertebrate rhodopsin is presented in Fig. I. There is a fair degree of agreement on most aspects of this sequence. In large part this is because the detergent selected by Tansley in 1931 [58], digitonin, yields an extracted rhodopsin which behaves very much like the rhodopsin in the intact rod outer segment. One area of concern has been the direct hydrolysis of Meta 11380 to retinal or its thermal decay through Meta I11465 (pararhodopsin). In their paper on the discovery of Meta 11380 Matthews et al. [20] suggested that the 465-nm compound was a cis-isomer side product. However, they did not study the 465-nm product in any detail. In a later paper by Ostroy et al [21], which presented data in agreement with Matthews et al. on most points, this 465-nm product was studied in more detail. Illumination of the 465-nm product produced rhodopsin, and it was observed as a major product under a number of temperature and pH conditions. Therefore it was concluded that Meta 111465 was in the direct thermal pathway for rhodopsin photolysis. Moreover, the 465-nm product seemed coincident with the main product of rhodopsin photolysis observed by Lythgoe and Quilliam [59], called Transient Orange. As the work on the rhodopsin sequence proceeded from detergent extracts to intact systems, the importance of Meta 111465 became even more apparent. In most cases studied, the Meta 11380 to Meta 111465 reaction was found to be a dominant step in the rhodopsin photolysis [23, 24, 29, 37] though both the pathway through Meta 111465 and the direct hydrolysis of Meta 11380 were usually assumed. While the direct hydrolysis of Meta 11380 is often emphasized, that pathway is not a major process under most conditions. Baumann's results [24, 29] indicate that the direct hydrolysis is 27 i~i in the frog (21'~C) and 25 7J~,in man (36°C), with a smaller percentage at lower temperatures. The maximum bypass yet found for rhodopsin is 50~o by Brin and Ripps [60] at 22"C in the skate, with reduced amounts at lower temperatures. No meta 111405 was observed in the sequence for the prophyropsin of the carp retina [61 ]. In our temperature analysis of the thermal decay of Meta II38o in the frog

97 retina [37] we did find two somewhat different reactions. The slowest process had the characteristics of the Meta II38o to Meta III465 reaction with a Q~0 of 2.5, enthalpy of activation (AH*) of 17 kcal/mol, entropy of activation (/IS*) of --15 cal/degree per mol and no pH dependence. The other reaction had a Q~o of 7.3, ,IH* 35 kcal/mol, AS* -- + 5 4 eal/degree per tool and a substantial pH dependence. Because of the values of the activation parameters we have recently assumed that the latter reaction is the Meta II38o to Meta I478 reaction (See Fig. 3), but it could represent Meta [I38o hydrolysis. Even if it were the case, that pathway would still represent only 17 j% of the total at 23°C. It is very difficult to interpret whole retina spectral changes with certainty. A number of processes with multiple reaction rates [30, 31, 34, 37], overlapping spectra, and different temperature dependences [21] are occurring simultaneously. For example, the thermal decay of Meta II38o has a smaller temperature dependence than the thermal decay of Meta II[46s [21]. Thus at the higher temperatures the thermal decay of Meta II38o would be the slower process and no Meta III465 would be observed even if it were the major product. Because studies at a variety of temperatures give the additional parameters of Q~o, enthalpies of activation and entropies of activation (which appear to be characteristic of the reactions) it would be useful if more such studies were presented. With respect to other intermediates, the low temperature intermediates bathorhodopsin543 (previously prelumi543) and lumirhodopsin497 have now apparently been observed at physiological temperatures [32, 33, 35]. This is discussed more fully in the next section (C1). Also a new low-temperature product, hypsorhosopsin43o, has been obtained by illumination at --268°C with wavelengths greater than 530 nm [26]. Excellent discussions of the possible retinal configurations involved in native rhodopsin and these low temperature products are presented in a recent a~ticle by Wald [25] and an earlier review by Abrahamson and Fager [27].

IlC. Conformation changes of rhodopsin during photolysis HC-I. General changes. There have been a number of studies which provide data on the conformation of the native vertebrate rhodopsin and the changes that occur upon illumination. They are summarized in Fig. 3 for detergent extracts of the protein. The relation between the detergent studies and studies in the intact system are discussed in section C2. In the native rhodopsin the binding site appears to be well protected. It does not react with the Schiff base reducing agent, sodium borohydride [69], and reacts only slowly with cyanoborohydride [70]. Also the rhodopsin spectrum shows no major effects over the pH range from 4 to 8 [68]. In the native state, the binding site appears to be a protonated Schiff base bond [53, 69, 70]. The protein has approximately 54 titratable acid-base groups [68] and two sulfhydryl groups are reactable in the native state [78]. The differing rates of reaction of the sulfhydryl groups suggest that they are in different regions of the protein [72]. Upon illumination a number of spectral and conformational changes occur. The bathorhodopsin543 to lumirhodopsin497 reaction has small activation parameters

23(2nd order) 25.2(R08 - 4 0 - 5 0 ° C )

18

+60

12.5 (ROS)

I0

~H*

(kcal/mole)

Ls*

(ROS)

-10 (Rat retina)

2 (Rat retina)

16 (Rat retina)

19 (Rat retina)

.~'

l

48

30 (Rat retina)

37

32 (Rat retina)

17 (Frog retina) -15 (Frog retina) ~--~ Meta II_~ (direct hvdro!ysis) ' ~0 35 (Frog retina) 5 4 (Frog retina)

-7

19

O

-50 (pH 5.1)

7.5

20 (Frog retina)

19 (Frog retina

O

91 (Rat retina)

41 (Rat retina)

54 (Rabblt retina)

70 (ROS)

31 (Rabbit retina)

~

28 (DDAO)

(ROS)

-5.8 (SOS +3 t o +i~°C) (2nd order)

21.7 (2(!8 +3 t c *180()

/,5 (EOS - 4 0 to -50°C)

2-4 +49 (2nd order kinetics)

+160

12-18

-2 to +5

(e.u.)

19 (DDAO)

35

o

o.--O~

@

4.5(ROS +3"!8eC) ~--~ 3.5(ROS +3+18 o C) ~ ~_ (2nd order)

~m~-. O~

| ~

7"

o~

~_

O ~

~'~

<

.~j

g .~"

>-~

Activation Parameters Bovine digitonin soln. unless stated otherwise (Ref$. 20-23, 27, 30, 31, 34, 36, 37, 62-67)

[

no ionization changes pB < 5.4

•

L--.~Retina1387

+ Opsin

1

_H+ ~ NRO440~h~0365 +H

"~'-"

111465

II380 q

1478

(Pararhodopsin465)

Metarhodopsin

(pK ~6) (pH 5.4-7.7)

-H +

.__ Zetarhodopsin

Metarhodopsin

Ill

Lumirhodops in497

binding site

54 Titratable groups (34 base; 20 acid) Spectrum insensitive pH 4-8

Protonated binding site Cyanoborohydrlde will react with binding site after 24 hr., 25°C

to

66 titratable groups (41 base, 25 acid).

--Phosphorylation of rhodopsin (or possibly at an earlier sta~e) Possible chromophore transfer to another amino ~rcup. Pr~tonated binding site -H p B > 7.7

2 SH groups exposed simultaneously same rate constant - corresponding thermal decay of Neta II380

Sodium borohydride reacts with binding site Unprotcnated bindinc site

Protonated

Hypsorhodopsin430

2 SH groups tltratable Different rate constants

(Pre- lumirhodops in543 )

Bathorhodopsin543

Rhodopsin498 q

oo

99 s,aggesting only minor protein conformation changes. The very rapid speed of that reaction with a half-life of 30-50.10 -9 S (25°C, Fig. 1) is also consistent with this notion. The activation parameters for the lumirhodopsin,97 to metarhodopsin [478 reaction are quite variable (Fig. 3). This variability may result from a change in the nature of that reaction. Rapp et al. [27, 34] found that the temperature dependence of the rate constant was quite different in the --40 to --50°C range compared to the + 3 to + 18°C range. It may be that the intermediates isolated at the low temperatures are not the same as those observed at the higher temperatures. For the early intermediates, it is unfortunate that the data obtained at physiological temperatures often does not include a full spectra of the intermediate, or kinetic data as a function of temperature. If that data were presented then one could compare the wavelength of maximum absorbance, Q~o values, and activation parameters, with those obtained at lower temperatures to decide if one is observing the same product changes. Major conformation changes appear to occur during the thermal decay of metarhodopsin 1478 to Meta 11380. It is only at the Meta I1380 stage that sodium borohydride will react with the binding site [69]. Meta I478 and Meta I1380 are in a pH-dependent equilibrium with acid, high temperature and glycerol favoring Meta 11380 [20,21] (Fig. 4a). The group p~( associated with the equilibrium is approx. 6.4 (refs. 20 and 24 and Fig. 43) and that group is not available prior to the Meta I478 stage (ref. 71 and Fig. 4b). Although the group involved in the equilibrium must be protonated to produce Meta I[38o (one proton per molecule is added [71]), the absorption and Raman spectra show that the binding site becomes deprotonated during this reaction [22,53,55]. An intramolecular proton transfer seems to be occurring [52,79]. Fig. 5 illustrates the changes in ionizable groups that are thought to occur during this reaction, and Fig. 4b shows the direct measurement of the proton uptake. Also, the reaction has activation parameters indicative of major conformation and charge disordering processes. The enthalpy of activation (3H*) is 19-41 kcal/mol and the entropy of activation (,dS*) is + 2 0 to + 9 0 cal/degree per mol. The proton uptake observed during this reaction is consistent with these activation values, since proton uptake would cause the protein to become less negatively charged and result in a disordering of the surrounding water. Conformation changes are also evident during the thermal decay of metarhodopsin 11380. The thermal decay of Meta 11380 directly to retinal involves hydrolysis of the binding site and separation of the retinal and opsin. Rotmans et al. [74] have presented data which suggests that the chromophore binding site may be transferring from one lysine to another during the thermal decay of Meta 1138o. During the thermal decay of Meta 11380 to Meta III,65, two additional sulfhydryl groups of the protein become exposed simultaneously to reagent [72]. The spectral shift associated with the Meta I1380 to Meta 111465 reaction indicates a re-protonation of the binding site but the protein releases protons during that reaction (Fig. 4c). Again an intramolecular proton transfer seems to be occurring, though in the opposite direction (refs. 52 and 79, Fig. 5). The data is consistent with a conformational disordering and

100 charge ordering during the Meta 11380 to Meta I[[465 reaction. The conformational disordering is indicated by the exposure of the sulfhydryl groups. The charge reordering is caused by the loss of protons because that will increase the negative charge on the protein and align the surrounding water. The negative (or small positive) entropy of activation of this reaction (AS* -- --50 to ÷ 2 cal/degree per tool) and small enthalpy of activation (/IH* = 7.5 to 19 kcal/mol) are indicative of these effects. Unlike the Meta 1,78 to Meta I[38o reaction which requires protonation of an ionizable group for it to proceed, the thermal decay of Meta l13so will occur independent of the pH. However, the state of those ionizable groups appear to determine the products that are formed [52]. In the pH range from 5.4 to 7.7 some ionizable group (or groups) release protons and Meta I138o thermally decays to Meta 11146s. Below pH 5.4 no proton changes are detected in solution and Meta II~so thermally decays to the protonated Schiff base N-retinylidine opsin~no. The spectral changes indicate a re-protonation of the chromophore-binding site during this reaction (see Fig. 5). At pH values above 7.7 proton release is observed in solution and the Meta 1138o thermally decays to the unprotonated Schiff base N-retinylidine opsin365. Thus the simple base Schiff compounds can be observed directly at the pH extremes. A representation of the ionization changes that are thought to occur is presented in Fig. 5. Except at extreme pH values the hydrolysis to retinal3s7 occurs from Meta II38o or Meta llI46s. The hydrolysis represents a major chemical change and the activation parameters reflect this. The Meta 1138o to retinalas7 process has a probable enthalpy 1.0 I (a)

8.0

9.5

0.5 o

300

400 Wavelength

500

600

( nm )

Fig. 4. Hydrogen ion changes during rhodopsin photolysis. (From Wong and Ostroy [71] and Ostroy [52]). (a) Effect of pH on Meta I47s ~- Meta 1Iaso Proportions. Temperature 3°C. (Derived from data in ref. 52). (b) The Meta 1478 to Meta l13so Reaction. pHi 4.5, 150 /tM Bromocresol Green, 50%/, glycerol. Difference spectra between bleached and unbleached sample at

101 of activation (AH*) of 35 kcal/mol and an entropy of activation (AS*) of 54 cal/ degree per tool and the Meta I1146s to retinal387 process has an enthalpy of activation (AH*) of 32 to 37 kcal/mol and an entropy of activation (AS*) of + 3 0 to +48 cal/degree per mol (Fig. 3). The evidence for conformation changes during the vertebrate rhodopsin sequence seems quite extensive. However, it has been observed that only minor 0.3~-

(b)

0,2

0.1

~=o.1 @

.~ 0.2 .< 0.3

0.4

0.5 L

I 400

300

I 500

I 600

Wovelength

I

700

(nm)

0.02

(c)

E c

0.2

ko

o.2~

0.I °u c

c 0.1

//S/~/~

%'°'~o.~.~o.~

m

<

0o

do

3;0 Time

4 5' 0

600

(seconds)

the s a m e temperature. (1) - - 2 2 ° C (2) 15°C s h o w i n g build-up o f Meta llsso a n d s i m u l t a n e o u s p H increase at 615 rim. (3) - - 1 5 ° C (15 min. after 2) 0.96 mol of H + per tool M e t a I l s , o (c) p H and Meta Ilsso changes at 25°C. pH~ = 5.60. (,l) 380 n m ; (O) Bromocresol G r e e n at 6125 A. Arrow indicates e n d o f illumination.

102 NR044 o H -C=~-

-RH+ (pK ~6.4) -RH+ (pK ~6

Rhodopsin

Met a 1478

H

Meta 111465

H

H

-eft-R

Hera 11380

)

-c N(pK -3.5)

-RH+ (pK-9.5)

-R

(pK ~6.4)

-RH+ (pK ~6.4)

-RH+ (pK -6.4)

-RH+ (pK ~9.5)

-RH+ (pK ~9.5)

-R

(pK ~6

)

NR0365 -C=N-R

(pK ~ 6 . 4 )

-R

(pK ~6

)

Fig. 5. Representation of the change in ionizable groups during the photolysis of vertebrate rhodopsin. (Taken in part from Ostroy [52] and Lewis ct al. [55]).

circular dichroism changes occur in the 220-nm region during the photolysis [80,81]. Since the circular dichroism in that region is most sensitive to (,-helix to random coil transitions, rather than other peptide chain rearrangements [82], it may be concluded that such ~z-helix transitions are not occurring. Major conformational changes may be occurring without any effect on the circular dichroism in that region. In fact, (t-helix to coil changes can be induced by heat denaturation of rhodopsin [83] (as it is induced in other systems [82,84]) with appropriate circular dichroism effects. Moreover, one would not expect a substantial denaturation during the rhodopsin photolysis since it is known that the addition of I l-cis retinal to a photolyzed rhodopsin sample can regenerate rhodopsin. It would be surprising if that occurred with a completely denatured protein. IIC-2. Comparisons of digitonin extracts and intact systems. One of the more fortunate aspects of the study of rhodopsin biochemistry has been the general use of the detergent digitonin in its solubilization. The close correspondence in most properties between digitonin extracts and intact systems are quite striking. The main thermal intermediates and the activation parameters which are indicative of the protein conformation changes, are generally the same in the two systems (Figs. l, 3). As studied by kamola et al. [85] even the equilibrium constant for the Meta I~v8 Meta Iis8o reaction and its response to pressure are the same in the two systems. This

103 is not true of extracts with emulphogene [85], lauryldimethylamine oxide [85], or dodecyldimethylamine oxide [67]. Also digitonin extracts regenerate to rhodopsin upon the addition of l l-cis retinal, as do pigments in rod outer segments [86] or in intact retina [87]. However, this is not true of emulphogene [86], Triton X-100 [88], cetyltrimethylammonium bromide [89], or lauryldimethylamine oxide [85] extracts. This property of the digitonin extracts may be related to its ability to retain more of the lipid components. Shichi [86,90] has shown the importance of lipid in the regeneration of rhodopsin. The loss of regenerability caused by aging, petroleum ether extraction, or phospholipase A treatment could be reversed by lipid addition. One of the few differences between digitonin extracts and rod outer segments is the absolute rates of the various reactions. They are usually faster in the digitonin extracts (Fig. 1). To explain the differences in the rates of reaction, attention has been focused on the lipid components of the rhodopsin complex. Williams et al. [91,92] have investigated the effects of lipids on the rate constants for the production of Meta lI3so. The addition of phosphatidylethanolamine to an aged preparation of frog rhodopsin in digitonin restored the original rate constant for the production of Meta II38o. Phospholipid effects on rate constants have also been observed in dodecyldimethylamine oxide [67]. Thus the more native behavior of digitonin extracts compared to other detergents may be associated with its ability to retain the lipid components, though nonhomogeneous dissolution [93] or loss of phospholipid [86, 90] can affect these properties. Chemical changes of the photoreceptor cell may also be affecting the rates of the rhodopsin reactions. Donner and his coworkers [94,95] have reported that external calcium concentrations and the percent of bleaching affect the rate of decay of the rhodopsin photoproducts in the isolated/iog retina. Some workers attribute the multiple first-order forms to the digitonin extracts, pointing out that they do not obtain such multiple rates in rod outer segments [67, 93]. However, other workers have obtained such multiple first order rates in rod outer segments and retinas [22,31,37,63,96]. Williams et al. [91,92] found that the lipid content affected the number and relative percentage of fast and slow first order reactions involved in Meta II38o production. However, under all conditions, multiple first order rates were observed. There are, of course, some properties of the intact rod outer segment that cannot be duplicated in detergent extracts of rhodopsin. These include properties that require the membrane or enzyme organization of the rod outer segment such as the light activated phosphodiesterase (Section VI), the rhodopsin phosphorylation (Section IIC3), or even rhodopsin proton pumping (Section VIII). The situation with the light-induced exposure of the sulfhydryl groups is not clear. Some workers have reported that they could react with certain sulfhydryl groups of the rhodopsin in the dark but were not able to observe the exposure of additional groups on illumination [97,98], whereas others have indicated that they were not able to react with any sulfhydryl groups of rhodopsin in the dark but were able to react with one sulfhydryl group after-illumination [99]. These data may be indicative of the problems associated with reacting a water-soluble reagent with functional groups in the membrane.

104

IIC-3. The phosphorylation of vertebrate rhodopsin. The phosphorylation of proteins is a mechanism known to cause enzyme activation (as in the case of phosphorylase b [100]) or protein conformation changes (as in the (Na +, K+)-activated ATPase [101]) or ion conductance changes (as in the synapses of the superior cervical ganglia [102]). (Also see section VI). Thus in the visual system even though the purpose of the rhodopsin phosphorylation is not yet known, its existence would suggest a possibly important role. First Kuhn and Dreyer [103] and Bownds et al. [[04], and later Frank et al. [73] reported the phosphorylation of rhodopsin in suspensions of rod outer segments at 30-37°C. The major characteristics of this phosphorylation are: (a) It takes place in the dark after rhodopsin illumination [73, 103-107]. (b) The kinase activated by the light is specific for the rhodopsin [105,107-109]. (c) For the frog, illumination of a small fraction of the rhodopsin is sufficient to phosphorylate a large fraction of the total rhodopsin, on the order of 10-50 rhodopsins phosphorylated per rhodopsin bleached [104]. In cattle, only one rhodopsin may be phosphorylated per rhodopsin bleached though larger numbers (approx. 5-6) are attainable [73,105,110]. (d) ATP is required and the phosphorylation can occur with extracellularly added ATP [73,[03,104]. (e) Mg 2 ~ is required, Ca 2+ is slightly inhibitory, and no effect is observed with Na +, K +, cAMP or ouabain [73,106,108]. (f) It may be slow with half-times of 2 rain in frog (21°C) and I-2 rain in cattle (36°C) [75]. However, some workers have suggested that the phosphorylation may be rapid because much of the phosphorylation had already occurred when they measured the phosphorylation within 15 s of the initial illumination [77], and altering

Q

.c 1.0

Q

0

3 0.8 5 E --.. 0.6

ff

0.4

8 C 13-

9

0.2 d 10

20 30 Min of incubation

40

50

Fig. 6. Phosphorylation of frog rod outer segment membranes. ( F r o m Bownds et al. [104]). (a) C o n t i n u o u s illumination bleaching 3 ~ of the rhodopsin per min. (b) 1 - 2 ~ bleach prior to [~2P]ATP addition. (c) 0.6% bleach. (d) 0.1% bleach. (e) Dark.

105 the rate of formation of the slow rhodopsin photoproduets had no effect on the phosphorylation [75,76]. (g) The phosphorylation occurs in vivo [107,111] as does the dephosphorylation [75,107,111]. (h) The phosphorylation may decrease the sensitivity of the photoreceptor. The time-course of dephosphorylation appears consistent with the time-course of dark adaptation [107] and phosphorylation inhibitors (such as adenosine) increase cell sensitivity at low light levels [112]. Fig. 6 illustrates some of the characteristics associated with rhodopsin phosphorylation. The characteristics of the phosphorylation are most suggestive of an adaptation or regeneration function in the photoreceptor. However, the fastest speed of the phosphorylation has not yet been determined and it is possible that it could occur on the time scale of transduction. HD. Rhodopsin as a membrane protein IID-1. Location. There are a number of rhodopsin properties that are related to its role as a membrane protein. It is not clear if these properties play an active role in the process of vision, but they are some of the structural parameters of the system and are not found in any single place in the literature. The frog rod outer segment has a diameter of 5-7 # m and a length of 35-50/~m with approximately 109 rhodopsin molecules per rod [38,113]. Since there are approximately 1800 disks per outer segment [114] the rhodopsin concentration is 1.67 • 106 molecules per disk. With the outer segment volume approximately 1 • 10-12 1, the calculated average rhodopsin concentration is 2 mM (measured as 2.5 mM, ref. 38). The bovine rod outer segment is smaller with a diameter of 2 #m, length of 7-10 # m [115], and a volume of 2.7 • l0 ~4 1. There are l06 rhodopsins per outer segment [116] and 450 disks per outer segment, yielding an average rhodopsin concentration of 0.06 mM. The human rod outer segment is similar with a 2-/zm diameter, 40-60/~m length, 10 v rhodopsins per outer segment [117], 1000 disks per outer segment [114], a volume of approximately 1.6 • 10-13 1 and average rhodopsin concentration of 0.1 mM. The Drosophila rhabdomere is 1.2 # m in diameter and 60 # m in length and the ommatidium is 17 # m in diameter and 70-125/tm in length [116]. In the Limulus ventral eye the rhabdomere length is 180/~m and the ommatidium is 180 # m in diameter 350 # m in length [12]. In Limulus there are 5 • 105 microvilli per rhabdomere [12]. X-ray analysis, electron microscopy and specific chemical reactions have been used to locate the vertebrate rhodopsin in the disk membrane. The clearest data have been the chemical reaction data. Using a variety of enzymes, Bonting et al. [118], Saari [119] and Trayhurn et al. [120] have removed a 10 000-15 000-dalton subunit from the rhodopsin in the disk membrane. Also Steinemann and Stryer [121] reacted concanvalin A with a carbohydrate moiety of rhodopsin in the disk membrane. Dratz et al. [122,123] caused the fluorescenl dye FITC (fluorescein isothiocyanate) to react with rhodopsin in the disk membrane after sonically treating their preparation.

106

Thus some part of the rhodopsin is exposed on the external surface of the disk membrane. Although proteins have been found which extend completely across membranes, in the visual system no clear data yet exist to suggest such a situation. X-ray analysis seems inadequate for locating the position of the rhodopsin in the disk membrane since the reports on the rhodopsin location have been mixed [124-127]. Electron microscope data have also resulted in conflicting statements about the rhodopsin location [128,129]. Some of the difficulties associated with interpreting freeze fracture electron microscope data in myelin [130] may be applicable to the rhodopsin problem. If the conditions are favorable, however, it seems possible that the electron microscopic analysis of Henderson and Unwin (ref. 131 and Fig. 12) used for the Halobacterium halobium could be applied to the vertebrate rod system. Rhodopsin may also be contained in the plasma membrane of the rod outer segment. Both electrophysiological and structural data have suggested such a situation [132135]. HD-2. Morement o['rhodop,s#~. Some efforts have been made to determine the freedom of movement of rhodopsin in the disk membrane. It has been known for some time that the rhodopsin chromophore has a preferred orientation in the plane of the disk membrane. Dichroic ratios of 4 : 1 are not unusual [136]. By measuring the dichroic ratios after bleaching Harosi [137] has shown that this orientation is preserved up to the Meta !II4o5 stage in the bleaching process. In spite of this restriction, data have been obtained which indicate that rhodopsin may be free to move in the other directions: to rotate on its own axis perpendicular to the disk membrane, to move laterally in the membrane, and perhaps to move in or out of the membrane. Cone [35] followed the dichroic ratio at 580 nm in isolated frog retinas in response to illumination with polarized light. A rapid rise in dichroism followed by a rapid decay (t2°/c " = 3.0 ! 1.5 ¢ts) was observed. Since under the same conditions the decay of bathorhodopsins43 and lumirhodopsin497 have somewhat different half lives ,tr 2°°Cl/2of 0.17 (+0.03) /~s and 50 ( 5 20) /~s respectively), it was concluded that the decay is caused by native rhodopsins which are free to rotate on their axis in the membrane. An experiment showing lateral movement of the rhodopsin was reported by Poo and Cone [138] and Liebman and Entine [139]. By bleaching one half of the disk they were able to observe the apparent movement of the unbleached rhodopsin into the bleached area with a half-life of approximately 30 s. The data do appear to show rotational and lateral movement of the rhodopsin, though multiple rates of decay for the intermediates, rhodopsin regeneration, or long lasting intermediate products, could interfere with the interpretation. There is some question about whether rhodopsin moves in response to light. in an X-ray study Blasie [I 24] reported the sinking of rhodopsin further into the disk membrane. From its pH dependence he concluded that the basis of this process was negatively charged rhodopsin becoming less negatively charged. (The suggested charge changes are consistent with the charge changes that occur in rhodopsin (Figs. 4c, 5)). Using electron microscopy, Mason et al. [129] observed the light-induced movement of rhodopsin from the internal surface of the disk membrane towards

107 its center. However, Chabre [126] was not able to detect any movement of the rhodopsin in response to light. Schwartz, Dratz and Blasie [140], in recent reports, have observed some changes in their X-ray pattern in response to light, but have suggested that it represent only a minor membrane rearrangement rather than a movement of the rhodopsin. The present prevailing opinion is that rhodopsin does not move in response to light.

III. RHODOPSIN CHANGES AND RETINA ELECTRICAL SIGNALS Some basic studies have been completed on the correlation of rhodopsin changes and visual receptor potentials. In the vertebrate rod, Cone [141] in the albino rat and later Gedney et al. [142] in the frog, showed that the time course of the R2 part of the early receptor potential corresponded with the time scale of the metarhodopsin 1,78 to II38o reaction. These data are shown in Fig. 7. Since the major reactions in the thermal decay of metarhodopsin II38o are slow (Fig. 1), and the late receptor potential follows the R2 phase of the early receptor potential by 5-55 ms [143], the above data indicates that no rhodopsin intermediate process corresponds directly with the late receptor potential. The results therefore imply that the late receptor potential is initiated by some delayed chemical process from earlier changes of rhodopsin. The metarhodopsin I478 to metarhodopsin l13so reaction, as suggested previously by Abrahamson and Ostroy [22], is the most likely candidate for this initial rhodopsin process. It is the dominant rapi0oreaction observed in any retina and involves major conformation changes (Fig. 3). The conclusion from the preceding data that the transduction mechanism involves some added processes after the crucial change of rhodopsin has been prevalent for some time. Its main basis had been the structure of the vertebrate rod. Since the rhodopsin is contained almost exclusively in free floating disks [144], to alter the permeability of the plasma membrane some chemical message from the disks seemed essential. Another basis for this conclusion has been the great sensitivity of the rod cell. Thus some multiplying mechanism seemed necessary if the absorption of a single photon by rhodopsin were to be responsible for closing 10 1 000 sodium pores of the plasma membrane [145]. In the vertebrate retina a number of simultaneous studies of rhodopsin changes and adaptation processes (i.e. the reduction in amplitude and sensitivity after stimulation) have been completed. There is some agreement that the total rhodopsin concentration is one factor in the adaptation level [146,147]. Direct evidence for this has recently been obtained by Pepperberg et al. [87]. By adding 1 l-cis or 9-cis retinal to a retina in which 90 °/., of the rhodopsin had been bleached, they were able to obtain a recovery of receptor potential amplitude and sensitivity, coincident with the regeneration of the rhodopsin. The role of the photoproducts of rhodopsin in photoreceptor cell processes has been difficult to prove, because there are so many slow rhodopsin reactions and products (See Fig. 1), and adaptation is such a complex process. Donner artd Reuter [148] showed similar time-courses between decreased concentrations of

108

RI 0. i

LRP I

R2 ERP

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R2

t

R2 (formoldehyde)

b. t.J 0.3ms -"%A 483

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AA 404

6"A 3 8 0 n m C.

18"C

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Fig. 7. Correlation of the Meta 1,,78 to Meta 1138o reaction and the early receptor potential. (From Cone [141] and Gedney et al. [142]. (a) Extracellularly recorded receptor pote.,ltial of the frog. The corenal positive (R~) and corneal negative (R2) componen~ of the early receptor potential (ERP) are followed by the late receptor potential (LRP). R2 : 340/~v LRP 100 #v. (b) Simultaneous recording of the time course of the early receptor potential and absorbance changes in the rat retina. One eye soaked in 1% formaldehyde at neutral pH for 3 h. Response amplitudes R~ 140/tv, R2 600 /tv, R2 (formaldehyde) 200/tv. Approximate change in absorbance 483 nm, 0.005; 405 n:11, 0.02 absorbance units. (c) Rhosopsin ERP and transmittance change at 380 nm (build-up of Meta 11~8o) in the frog retina. Comparison of inverted ERP and transmittance change at 380 nm from numerous expe,iments. Absorbance changes 0.1-0.3. Peaks are scaled to sample amplitude.

Meta 1138o and cell threshold in the frog retina and concluded that Meta i138 o may control rod sensitivity. However, Weinstein et al. [149] a n d F r a n k and Dowling [150] showed that the rat b-wave (corneal positive portion of the electroretinogram) had properties which could not be correlated with p h o t o p r o d u c t processes. In an intracellular study, G r a b o w s k i a n d Pak [96] studied the thermal decay of Meta l13s o and threshold properties of the receptor cell in the axolotl retina. While the two processes appeared to have similar kinetics, the correspondence was not exact enough for them to conclude that the two processes were interdependent. Ernst a n d K e m p [151] studied the aspartate isolated receptor response a n d p h o t o p r o d u c t changes in the rat retina as a function of temperature. Their data shows a r e m a r k a b l y close correspondence between the time-course of recovery of the receptor response and thermal decay of Meta 111465 at 24, 29 and 34°C. Other studies

109 intended to relate rhodopsin changes to the late receptor potential have generally centered on kinetic analysis of the time course of the late receptor potential as a series of first order reactions [152,153]. Though the models obtained from such an analysis should prove quite useful in testing mechanisms for photoreceptor function, there are certain assumptions inherent in this approach that require great precaution in their interpretation. Though rhodopsin intermediate reactions are usually summarized as single first order reactions, a minimum of two and usually three such concurrent reactions for each process are found, even in rod outer segments or whole retinas (Fig. 1). Also, even if the late receptor is initiated by a single first order rhodopsin reaction, it is not clear that the subsequent conductance changes and membrane potential changes of the photoreceptor cell can then be described by first order kinetics or a sequence of first order reactions. Finally, recent data on receptorreceptor interactions in Bufo marinus [154] indicate that these receptors are electrically coupled. Depending on the type of illumination this may lead to some complexities in the kinetics of the intracellular response. In invertebrate systems the less complex rhodopsin reactions should simplify the correlation between the rhodopsin and the electrical changes of the photoreceptor. For example, in the Drosophila we know that the main rhodopsin reaction is the transition from a rhodopsin48o to a long-lived metarhodopsins8o [39,155]. The conversion of rhodopsinc8o to metarhodopsinsao will produce a receptor potential. This is true even when the wavelength of the light stimulus is selected so that there is a steady-state concentration of the rhodopsin48o and metarhodopsins8o [13]. Since a steady-state concentration of rhodopsin~8o and metarhodopsins~o are produced under these conditions, the data implies that the sodium conductance is not altered directly by the conformation states represented by rhodopsin48o or metarhodopsin58o. It suggests that the conversion from rhodopsin48o to metarhodopsins8o initiates other steps which result in the conductance change.

IV. HYDROGEN ION EFFECTS OF THE PHOTORECEPTOR CELL pH gradients and proton transfer are thought to be the major mechanism underlying the process ot energy transduction in chloroplast and mitochondria [156]. The main function of bacteriorhodopsin is thought to be proton pumping (ref. 49 and Section VIII). pH changes of the medium or changes in the ionization of membrane proteins can alter the permeability or binding characteristics of neuronal membranes [157] and hydrogen-calcium competition is well documented [158]. Although in the vertebrate or invertebrate photoreceptor, a specific functional role for hydrogen ion or ionization changes has not yet been shown, the system exhibits many of the characteristics necessary for such a role. Vertebrate rhodopsin undergoes a series of ionization changes during its photolysis cycle (Figs. 4 and 5, refs. 52, 65, 68 and 71). Illumination-induced pH changes have been observed in the barnacle photoreceptors [159]

110 and in the perfusate of vertebrate rod outer segment suspensions [160-162] and vertebrate retinas [163]. It would seem that the dipole [141[ or ionization [164] changes of vertebrate rhodopsin may be responsible for the early receptor potential. Cone [141] suggested that the early receptor potential was caused by oriented rhodopsin dipole changes. The early receptor potential is a fast, mainly capacitive [165,166] response which corresponds to the metarhodopsin l~Ts to metarhodopsin [138o reaction, and is eliminated by a 10-min heating at 50~C (which disorients the membrane) [167]. Ostroy [164] suggested that the ionization changes of the rhodopsin may be responsible for the early receptor potential. An electrical potential that corresponded to the Meta 14v8 to Meta 1138o reaction was elicited from solution extracts of rhodopsin contained in a dialysis membrane system [164]. Since those signals could be eliminated in 0.1 M salt they appeared to result from a Gibbs-Donnan potential caused by the charge change of the rhodopsin. (The Photoproduct Early Receptor Potential responses of Meta 1478 and Meta 111465 are of the opposite sign to that of Meta I138o [168], consistent with their relative chalge changes (Fig. 5)). Hydrogen ion or pH effectors alter the vertebrate late receptor potential. Increased pH [163, 169], increased buffer concentration [163], or substitution of 2H20 for H20 [163] cause increases in the extracellularly measured late receptor potential amplitude. In Bufo marinus, Pinto and Ostroy [170,171] have recently altered the pH of the perfusate while intracellularly measuring the photoreceptor cell characteristics. A change in the extracellular pH from 6.8 to 8.0 or 7.3 to 7.8 (pK 7.6) (also ref. 172) caused a byperpolarization of the cell membrane potential (2-3 my), a decrease in the amplitude of the light-induced receptor potential, a lengthening of its time course, and a decrease in cell membrane resistance. Moreover, this resistance change could be obtained in sodium-free or chloride-free solution. The results indicate that the effects observed resulted from an increase in the potassium conductance. The results would suggest that pH changes of the photoreceptor are not involved directly in the sodium conductance changes of the plasma membrane. However, if the disk membrane exhibits the same permeability characteristics as the plasma membrane then a mechanism involving potassium conductance changes or rhodopsin ionization changes at the disk level can be speculated about. In the invertebrate, Brown and Meech [173] have shown that changes in internal pH (7.4. to about 6.0) decrease the potassium conductance of the barnacle photoreceptor. Also using a pH microelectrode Brown et al. [159] have recently shown that light induces a 0.5 pH unit decrease (from 7.4 to 6.9) in the barnacle photoreceptor. In Limulus, Coles and Brown [174] injected a series of buffers into the retinula cell. They found no major effect on receptor potential amplitude but did find that increased buffer concentration caused a decreased latency of the response. The data would appear to exclude a role for hydrogen ion as the transmitter substance of the photoreceptor cell. The data, however, may suggest a possible transduction role for rhodopsin ionization change in the receptor process. Depending on the original buffering of the cell, increased buffer concentrations would be expected to reduce the

111

internal pH change while improving the ionization of the rhodopsin. If a critical degree of ionization is needed to produce the receptor potential, under conditions of increased buffer concentration, the critical degree of ionization would be attained more rapidly, thus decreasing the signal latency.

V. C A L C I U M E F F E C T S O F T H E P H O T O R E C E P T O R

CELL

VA. Introduction The effects of calcium and cyclic nucleotides will be discussed in the next two sections. They are not direct properties of the rhodopsin but appear to be involved in the transduction or adaptation processes. The major evidence for the role of calcium has been obtained in three types of studies: (1) The dark current and photocurrent studies of Hagins and Yoshikami [175-177] in the rat. They used ion changes and specific ionophores to show that calcium mimicked the effect of light and to suggest that calcium might be the internal transmitter in the vertebrate; (12) the voltage clamp studies by H. M. Brown et al. [178] in the barnacle, showing that calcium can suppress the sodium current and that calcium permeability may have increased on illumination and suppressed the sodium permeability increase, and (3) the work of J. E. Brown and his colleagues [8-1 I ] in the Limulus ventral eye and the barnacle using ion and chelate injection and luminescent dye measurements. They show that an increase in intracellular calcium concentration occurs after illumination and that the calcium concentration increase can be induced by increased sodium concentration. They also show that the internal calcium concentration can control the cell responsiveness. VB. Vertebrates By measuring the voltage drop along the length of the albino rat photoreceptor, Hagins et al. [3,179] presented data on the current paths of that cell. In the dark, an inward current in the outer segment and outward current in the inner segment were observed. The effect of illumination is to suppress these dark currents. The results were an independent proof that light causes a decreased sodium conductance [1,2] and thus, an increased membrane resistance [1] of the vertebrate outer segment. A similar reduction in dark current was achieved by raising the external calcium concentration from l0 -5 to 1.36 mM or from 1.36 to 20 mM [175-177]. Effects could also be obtained when external Ca z+ concentration was changed from 0.01 to 1.36 mM or from 1.36 to 5 mM. From this and other data Yoshikami and Hagins [180] made the suggestion that calcium is the internal transmitter of the photoreceptor. They proposed that the internal Ca 2÷ concentration is 10 -8 M in the dark, with 10-3 M inside the disk and 10 3 M external to the cell. Upon illumination the intra-disk and extracellular Ca 2+ enter the cytoplasmic space of the cell, raising the concentration to 10 7 M and closing the sodium channels [175,177]. Some clear calcium effects have been obtained in the vertebrate photoreceptor. Yoshikami and Hagins [176] observed the rate of rundown of the late receptor

112 potential in a ouabain treated retina (as an indicator of the sodium permeability) and showed that the rundown was five times slower at 5 mM Ca 2+ than at 1.36 mM Ca 2+. (We have obtained similar results over a range from 0.1 to 5 mM Ca 2+ in the frog [172].) Thus external concentrations of calcium do appear to affect the sodium permeability of the rod photoreceptor. The internal [Ca 2+] seemed to be the important factor. Hagins and Yoshikami [177] added the calcium ionophore X537A and observed that current changes induced by a shift from 10-5 M to 2.10 -2 M Ca 2+ could now be obtained by a shift from 10 9 to 10 s M Ca 2+. In Bufo marinus Brown and Pinto [5] observed the effect of altered external calcium concentration on the membrane potential and photoresponse. Over the range from 0.6 to 3 mM external calcium, a proportional hyperpolarization and signal amplitude decrease were observed consistent with an effect of Ca 2+ on the sodium permeability of the outer segment. At higher concentrations, from 3 to 8 mM Ca/+, the response amplitude decreased more than expected if the only effect of calcium were on the sodium permeability. Perhaps the strongest evidence supporting the hypothesis that calcium is the internal transmitter of the photoreceptor has just been reported by Pinto and Brown [181]. They have injected calcium and the calcium chelator EGTA into the Bufo marinus outer segment. The injected calcium mimicked the effect of light in hyperpolarizing the outer segment. Immediately after the E G T A injection no light responses could be obtained when a small spot of light was used. Other calcium effects that have been found include: (1) An ATP-dependent accumulation of calcium by rod outer segments by Bownds et al. [182]; (2) the binding of calcium by the disks by Neufeld et al. [183]; (3) active Ca 2+ accumulation in disk membrane vesicles with calcium release and more active calcium accumulation upon illumination by Mason et al. [184] (the release was approximately 1 Ca 2+ per rhodopsin with less than 1 "J,, bleaching); (4) the release of 1-10 CaZ+/rhodopsin with rhodopsin bleaching of the order of 20~,, by Hendriks et al. [185], and (5) a lightinduced calcium release from artificial membranes containing rhodopsin by Hong and Hubbel [186]. Overall, in the vertebrate, external, and now internal Ca 2+ concentration changes, appear to affect the permeability of the photoreceptor in the correct manner for it to be an internal transmitter. Also Ca 2+ appears to be released from the disks, but it is not yet obvious that each light-activated rhodopsin causes the release of the 10-1000 calciums apparently needed for excitation [145]. One must be cautious in evaluating the data, since many of the calcium effects are over wide and unphysiological calcium concentrations and bleaching ranges.

VC. Invertebrates The larger and more easily penetrable cells of the invertebrate, and the application of some elegant techniques, have permitted more detailed experiments on the role of calcium to be carried out in invertebrate systems. Fig. 8 shows the currentvoltage relationship for the barnacle at a variety of calcium concentrations. In the low [Ca 2+] of 2 mM compared to the normal 20 mM of this preparation, the inward

113 xlO-eA 2

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40

mV _.__1_ 6O

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=

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Fig. 8. Current vs. voltage relationship in the barnacle photoreceptor as a function of calcium concentration. (From Brown et al. [178]). Light-initiated membrane current measured 5 ms after the onset of the light.

and outward currents are both increased. At the high [Ca2+] of 32 m M both currents are reduced. Thus Brown et al. [178] conclude that calcium's main effect is the suppression of the sodium membrane current. From the change in reversal potential, particularly in the absence of sodium, they also suggest that permeability changes to calcium may also be occurring. Recently, in a series of elegant experiments with the Limulus ventral eye and the barnacle, J. E. Brown and his colleagues have obtained considerable data on calcium effects. Initially Lisman and Brown [9] injected calcium or sodium into the photoreceptor cell and showed that both caused a decreased response to light proportional to the amount injected. From this data they suggested that the increased internal sodium concentration, caused by the light-induced sodium permeability increase, could increase the internal calcium concentration and reduce the cell responsiveness. Thus calcium would be involved in cell adaptation. This data is shown in Fig. 9. They have been able to show the calcium effects directly by following the calcium concentration changes of the cell with the calcium sensitive luminescent dye aequorin [8]. (Fig. 10). In the normal untreated cell the light-induced current per quantum decreases after intense illumination, and the decrease is accompanied by an aequorin transient. However, in a cell in which the calcium concentration is regulated with EGTA, after intense illumination no decrease in light-induced current per

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high Co~ -- I SO4 free

(b)

Fig. 9. Effect of calcium and sodium injection on the receptor potential of Limulus. (From Lisman and Brown [9]). (a) Intracellular iontophoretic injection of Ca 2+. (A) Stimulus intensity fixed; the magnitude of the injection current is marked below each injection interval. (B) Same cell as (A). Injection current was fixed at 5 hA; relative light 5 hA; relative light intensity is marked below each record. (b) Intracellular sodium injection and effect of calcium. (A) Na + injection of 4 10 nA for I rain of pipette filled with 1 M NaCI. (B) Injection of low-Ca 2+ (0.1 mM). No decrease in receptor potential is seen during or after sodium injection. (Star indicates 16-fold reduction in light intensity.) (C) Same as (A) except light intensity was 16 times brighter. (D) Na ÷ injection of 3 5 nA for 2 min. (E) Na + injection in high Ca 2+ (50 raM) producing extreme degradation of the light response. (F) Na + injection, intensity is 256 times less than D. Normal Ca 2+ is 10 raM.

quantum

[10,11] o r a e q u o r i n t r a n s i e n t [8] is o b s e r v e d .

Thus they have shown that

c a l c i u m c o n t r o l s cell a d a p t a t i o n in the Limulus v e n t r a l eye. B a d e r et al. [187] h a v e r e c e n t l y r e p o r t e d s i m i l a r results u s i n g c a l c i u m a n d E G T A i n j e c t i o n e x p e r i m e n t s in t h e h o n e y b e e . H o w e v e r , the m e c h a n i s m o f t r a n s d u c t i o n has b e e n s o m e w h a t m o r e elusive. W h i l e o n e w o u l d e x p e c t the processes o f t r a n s d u c t i o n a n d a d a p t a t i o n to be related, to d a t e the o n l y effect that m a y be r e l a t e d to t r a n s d u c t i o n is t h a t C a 2+ alters t h e l a t e n c y o f the p h o t o r e s p o n s e [188].

115

P

_ SM

:__

_

-,_ p?v

--~..t

$M'-a

PM S M --,

rnV

2s

.

PiM. S M "L

.. r

5°I

.

i

~

EL PMT

Fig. 10. Receptor potential and calcium changes in the barnacle photoreceptor. (From Brown and Blinks [8]). Aequorin responses and receptor potentials measured simultaneouslyfrom Balanus photoreceptors. Responses to (A) a brief stimulus and (B) a prolonged stimulus. (C, D) Another cell which was dark adapted for 30 min before (C) and 3 rain before (D). (E) Decay of aequorin luminescence from aequorin injected into artificial sea water V: membrane voltage. PMT: photomultiplier tube current. SM: stimulus monitor.

Vl. CYCLIC NUCLEOTIDE EFFECTS OF THE PHOTORECEPTOR CELL In a number of biological systems, cyclic nucleotides (often in conjunction with calcium) perform a variety of important functions. As a few examples, they have been implicated in the mechanism involved in the transmitter release from synapses, hormone release from glands, the hormonal effect at target organs, and the mechanism of action of transmitters on the postsynaptic membrane [189-191]. The general mechanism of action of the cyclic nucleotides as depicted by Rasmussen [189] is presented in Fig. 11. The main mechanisms involved are activation of protein kinases and control of internal calcium concentration. Thus, in the photoreceptor, even though the functional role of the cyclic nucleotides are not yet understood, they could play a major role in cell maintenance or function. In the initial studies of the cyclic nucleotides there were some difficulties because one could not be certain that one had pure rod outer segments and because the cyclic nucleotide system involved many components. However, there is now general agreement from a number of independent laboratories that the rod outer segments contain a light activated, ATP-dependent, phosphodiesterase which is 23 times more sensitive to cyclic G M P than cyclic A M P [192-198]. The change of activity is quite dramatic.

116

Ca2+

,

5"AM P

~

pp + 3t5LAMP • SPri

lW

[® IPrK ~,i,

X

\

Ca~+ mv

~_

\

!

.4

~,

,_

_

~'~/" ADP -20 7

Ca 2~.._...

~ o

---

SPra

Primary signal

Time

) ......i"

"\ -

~

-

4-- . . . . (E)

:'Y

Fig. I1. The basic aspects of the adenyl cyclase control mechanism. (From Rasmussen [189]). The basic aspects of the adenyl cyclase control mechanism. The primary signal which can he either chemical or electrical leads to membrane depolarization or entrainment of a series of action potentials (inset) with the activation of the enzyme adenyl cyclase (1, circled). This leads to an increase in cyclic 3', 5'-AMP and pyrophosphate (PP). The latter product is removed by the enzyme pyrophosphate (5). The increase in cyclic 3', 5'-AMP leads to an activation of a protein kinase (PrK) (2), an enzyme that leads to the phosphorylation of one or more enzymatic or contractile proteins (6) within the specific cell. Two key enzymes that regulate the concentration of cyclic 3', 5'-AMP and phosphorylated protein are phosphodiesterase (4), which catalyzes the hydrolysis of cyclic 3', 5'-AMP to 5'-AMP, and protein phosphate phosphatase, which dephosphorylates the phosphorylated or active form of the protein or proteins (6) within the cell. The second control element in the system is Ca 2+. An increase in the concentration of Ca 2+ in the cytoplasm is brought about by an increase in the uptake of calcium from the extracellular fluids or a mobilization of calcium from intracellular pools, or both. The mobilization of extracellular calcium is due to a direct effect of the stimulus on the plasma membrane of the cell, and the mobilization of intracellular calcium is brought about, with cyclic A M P acting on one or more intracellular membranes. The increased calcium within the cell serves one or possibly two functions. It is the specific activator of the phosphorylated protein (enzyme) (6) produced as a result of protein kinase (5) action. The increase in calcium may also activate other enzymatic reactions (8) within the cell, and it may act as a feedback inhibitor of further adenyl cyclase activation (1). The concentration of calcium within the cell is controlled not only by its leak into the cell, but by its active extrusion by a specific calcium 'pump' or membrane-bound calcium-activated adenosine triphosphatase (7) and by its energy-dependent calcium accumulation in one or more cell organelles (9). The protein (SPr,) phosphorylated by the protein kinase can be inactivated by a specific protein phosphorylase (3). The X and Y are substrate and product, respectively, of the reaction catalyzed by the active form of the enzyme SPr, phosphorylated by the protein kinase; Spq, the inactive protein substrate for the protein kinase; S and P are substrate and product, respectively, of other calcium-activated enzymes. The i and - - indicate positive and negative modification of the particular process, respectively.

K e i r n s et al. [ 197] r e p o r t e d a c h a n g e o f p h o s p h o d i e s t e r a s e a c t i v i t y f r o m a d a r k v a l u e o f 0.22 y m o l

cyclic G M P

hydrolyzed/min

p e r m g p r o t e i n t o 1.63 # m o l cyclic G M P

h y d r o l y z e d / m i n p e r m g p r o t e i n in t h e light. A b l e a c h i n g o f o n l y 1 % o f t h e r h o d o p s i n w a s sufficient f o r 98 % o f t h e c h a n g e in a c t i v i t y . T h e k i n a s e r e s p o n s i b l e f o r r h o d o p s i n

117 phosphorylation does not seem sensitive to the cyclic nucleotide changes [73, 106, 108]. The cyclic nucleotide changes have not yet been measured on the time scale of transduction events but they would appear to be slow compared to transduction [194,196, 199]. Since light acts to reduce the concentration of cyclic nucleotide by stimulating its enzymatic degradation, it is difficult to imagine that such a process could proceed rapidly and completely enough to be involved in transduction. Therefore, most of the work on the relation between the cyclic nueleotides and the visual receptor process has centered on the adaptation or maintenance of the photoreceptor. However, Ebrey and Hood [200,201 ] have noted no effect on the late receptor potential amplitude after exogenous addition of dibutryl cyclic AMP, cyclic AMP, or cyclic GMP, and no effect on adaptation upon the addition of dibutryl cyclic AMP. The only effect they have observed is a reduced late receptor potential amplitude after addition of some phosphodiesterase inhibitors, apparently caused by a reduction in the sodium gradient. In rod outer segment suspensions Bownds et al. [202] were able to recover sodium sensitivity more rapidly in the presence of added ATP, cyclic AMP, or dibutryl cyclic AMP. Perhaps the most dramatic results on the possible relation between cyclic nucleotides and photoreceptor functions are those reported by Lipton [203, 204] in Bufo m~rinus. Upon addition of the potent phosphodiesterase inhibitor, isobutylmethylxanthine, he obtained an initial increase in receptor sensitivity followed later by a prolongation of the light-induced intracellular response. The data is consistent with the cyclic nucleotide system acting to control the calcium concentration. Although the cyclic nucleotide system has properties that are very appealing chemically to provide for regulation, adaptation, or maintenance of the visual photoreceptor cell, the data are not yet sufficient to verify their involvement.

VII. THE PURPLE MEMBRANE OF HALOBACTERIUM HALOB1UM The purple membrane of the Halobacterium halobium has provided some exciting results that may relate to the functioning of both vision and photosynthesis. For vision the two most exciting results that have come from the work are: (l) that bacteriorhodopsin functions as a proton pump with a quantum yield of 0.7-1.0 protons per photon absorbed and a translocation of 200-500 protons per second at saturating light intensities [49, 205-207], and (2) that the bacteriorhodopsin is ordered across the purple membrane with seven closely packed a-helical segments perpendicular to the plane of the membrane [131]. This later picture which may be applicable to the vertebrate rhodopsin is presented in Fig. 12. (Also the vertebrate rhodopsin exhibits many proton and ionization changes (Figs. 4-6), and it could act as a proton pump, but no clear data on that paint has yet been obtained.) Like vertebrate rhodopsin, the bacteriorhodopsin has a retinal chromophore that is linked to the protein by a protonated Schiff base [48]. Also during the sequence of intermediates the chromophore deprotonates and then reprotonates. However, in many other respects the bacteriorhodopsin is not like the visual pigments. Rather

18

Fig. 12. A model of a single protein molecule in the purple membrane. (From Henderson and Unwin [I 31]). Viewed roughly parallel to the plane of the membrane. A model of a single protein molecule in the purple membrane, viewed roughly parallel to the plane of the membrane. The top and bottom of the model correspond to the parts of the protein in contact with the solvent, the rest being in contact with lipid. The most strongly tilted ~-helices are in the foreground.

t h a n l l-cis retinal, it has a 13-cis retinal or all-trans retinal as its c h r o m o p h o r e [45, 46,49]. N o isomerization occurs on i l l u m i n a t i o n [49]. Also it has a sequence o f spectrally distinct t h e r m a l intermediates t h a t seem similar to the vertebrate r h o d o p s i n products, but they are observed only at very low t e m p e r a t u r e s [44,45,49]. The intermediates and their properties are presented in Fig. 2. A t r o o m t e m p e r a t u r e the s a m p l e is actually stable to light. O t h e r c o n t r a s t i n g properties o f the b a c t e r i o r h o d o p sin include its low m o l e c u l a r weight ot 26000 [47] a n d its instability in detergent solutions [45]. It has been studied only as a p a r t o f the purple m e m b r a n e [44] or in p h o s p h o l i p i d vesicles [205]. D a t a indicating which properties o f the purple m e m b r a n e system are relevant to the functioning o f the visual system are yet to be obtained, but the d a t a are interesting a n d provide some testable hypotheses.

119 viii. HYPOTHESIS FOR VISUAL PHOTORECEPTOR FUNCTION To provide framework for the available data and to provide testable suggestions for future work a hypothesis for visual photoreceptor function is presented in Fig. 13. The hypothesis is based on the data and references which have already been presented. In the vertebrate it is assumed that it is the Meta I4,8 to Meta II38o transition of the rhodopsin that initiates vision. The charge change of the rhodopsin (or the localized hydrogen ion changes that occur) then causes an increase in the potassium permeability of the disk. This causes an increase in the calcium (or sodium) conductance of the disk. The increased calcium conductance would directly result in an increased cytoplasmic calcium concentration. If the system would operate through the sodium conductance change, it would initially cause an increased cytoplasmic sodium concentration which would then induce the release of calcium from membrane sites and increase the cytoplasmic calcium concentration. The phosphodiesterase activation at this time would cause the cyclic nucleotide system to be temporarily inactivated, thus permitting a larger increase in [Ca2+]. The calcium then reacts with the sodium channel of the plasma membrane and reduces the sodium conductance. The hyperpolarization of the photoreceptor cell causes an increase in the potassium conductance. To reverse the transduction process the rhodopsin proceeds to Meta IIl46s becoming more negatively charged and also phosphorylated. It would be more negatively charged than in the native state. This would serve to inactivate the rhodopsin and the disk in which the particular rhodopsin was contained. The cytoplasmic calcium would be returned to normal through the cyclic G M P and cyclic AMP system and the disk would be ready for new stimulation after the rhodopsin was dephosphorylated. For the invertebrate system a very similar mechanism is suggested. In that case the critical step is the rhodopsin to metarhodopsin transition. The charge changes are reversed from the vertebrate and the metarhodopsin is proposed to be more negatively charged and phosphorylated. Localized decreases in pH are indicated. In the external membrane these rhodopsin changes then cause a decreased K + conductance (decreased Ca 2+ conductance) and increased Na + conductance of the external membrane. For adaptation and recovery the higher internal sodium concentration would cause an increased [Ca2+]i. The calcium then reacts with the external membrane thus reducing the sodium conductance. As the cytoplasmic Na + concentration then returns to normal this (and perhaps the cyclic nucleotide system) would reduce the calcium concentration to normal. The rhodopsin would be prepared for another stimulus after dephosphorylation and regeneration.

ACKNOWLEDGEMENT I would like to thank Myrna Gaidos, Jan Martin, Steve Whistler and Meegan Wilson for their assistance and Drs. Clark Gedney, Lawrence Pinto and William Pak

1

|

LRP - - - ~! - . . . . .

1 I

m

EI

_ _

h,0

Meta I I 1 4 6 5

...............

I1380

Meta 1478

I

Rhodopsin498

Vertebrates

increased K + conductance

~[Ca + + ]

t pH)

disk can

,

I

c: ~' <:

z


~E

'

7~ ~, <

RP ----g ........................

Metarhodopsin580

hvll

Internal Na + concentration reduced. Ca +-+ concentration returns to normal regulated by Na + concentration and perhap~ cC~!P and c ~ . R)lodopsin dephosphor~'lation and regenerations.

~

L

Ca reacts with external membrane. Reduces Na + conductance.

l~creased Na + concentration causes increased concentration in cytoplasmic space. •

l

charged)

Ca ~q-

of external membrane

conductance

decreased K + conductance

Causes decreased Ca ++ Increased Na + conductance

Causes

(more negatively

.... d l n p K

Charge change of the rhodopsin Localized ~ pH Rhodopsin phosphor3/late d

C .... i n i o n i z a b l e g r o u p s a l

(Drosophila)

Fig. 13. Hypothesis for visual photoreceptor function.

After rhodopsln dephosphorylatlon again be activated.

Cytoplasmic Ca ++ concentration returns to normal regulated by cGMP and cdLMP (and perhaps Na + concentration)

Rhodopsin more negatively charged Rhodopsin pbosphorvlated K+ c o n d u c t a n c e of disk reduced below normal resting level, Ca 44" ( o r Na + ) c o n d u c t a n c e of disk reduced below normal.

+-+ + -Ca reacts with plasma membrane reducing Na con~actance. Hyperpolartzation causes increased potassium conductance.

[c~]

in

of disks

Causes increased Ca++ c o n c e n t r a t i o n cytoplasmic space. Phosphodiesterase activation ;[c~]

Causes increased Ca ++ ( o r Na + ) conductance of disks

Causes

P~hodopsin less negatively charged Intramolecular proton tramsfer (localized

Certain ionizable groups increased in pK (3.5 ~ 6.4)

Rhodopsin480

Invertebrates

121 f o r r e a d i n g t h e m a n u s c r i p t s a n d m a k i n g v a l u a b l e suggestions.

I w o u l d also like to

t h a n k t h e scientists w h o g e n e r o u s l y m a d e their figures a v a i l a b l e f o r i n c l u s i o n .

Sup-

p o r t e d in p a r t by N a t i o n a l Eye I n s t i t u t e g r a n t N o . E Y 0 0 4 1 3 a n d R e s e a r c h D e v e l o p m e n t A w a r d N o . EY32951 f r o m the U . S . P u b l i c H e a l t h Service.

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

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