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.+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 ~

~'~

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...
2MB Sizes 0 Downloads 0 Views