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A possible mechanism for the amplifier effect in the retina
Yision Res. Vol. 3, pp. 25-28,
Pergamon Press 1963. Printed in Great Britain.
A POSSIBLE
MECHANIST EFFECT
FOR
IN THE
THE
AMPLIFIER
RETINA’
THEODORE LOUIS JAHN Department of Zoology, University of Caiifornia, Los Angeles (Received 31 Augusr 1962)
transforms the 1I-cis-retinene of the rhodopsin molecule into all-transretinene, thereby making the molecule longer and highly resonant. It is proposed that these changes permit electron flow through the lamellar membranes, with the electrons provided by and received by oxidation-reduction enzymes, one on each side of a membrane. This conductive state of retinene occurs only in the lumi- and metarhodopsins. While conductive, a molecule of retinene, under electron pressure from oxidation-reduction enzymes, might conduct many electrons, thereby providing an amplifier mechanism, and causing depolariz&on of the membrane. Abstract-Light
R&urn&La lumi&re transforme le 11-cis&in&ne de la mol&ule de rhodopsine en transr&int?ne, ce qui rend la mol&ule plus longue et fortement r&onante. On propose que ces changements permettent le passage des tlectrons it travers les membranes lamellaires; il s’agirait des Clectrons lib&s ou cap& par les enzymes d’oxydo-tiduction, un de chaque c&t! de la membrane. Cet &tat de conduction du r&intne n’existerait que pour la lumi- et m&arhodopsine. Dam cet &at conducteur, et sous la pression des Bectrons provenant des enzymes d’oxyd*r&duction, la molt%ule de r&in&e pourrait conduire beaucoup d’&ectrons, se comportant ainsi comme un m&canisme ampiificateur et d&polar&ant la membrane. Z~rne~~~g-Licbt fiihrt das Cis-Retinen des Rhodopsinmolek~ls in Trans-Retinen ijber. Das MoIekSl wird dabei linger und in starkem Ausmasse resonanz~hig. Es wird angenom~, dass diese V~~nde~ngen einen Eiektronenstrom durch die lamellena~igen Membranen zulassen, wobei die E~ektronen van den ~yda~io~s-Reduktions-Enzymen abgegeben und a~genommen werden, die sich auf jeder Seite einer Membran befinden. Dieser leitende Z&and des Retinens tritt nur bei Lumi- und Metarhodopsjnen auf. Ein Retinenmojek~l kann so, wghrend es leitend ist, unter dem Elektronendruck der OxydationsReduktions-Enzyme viele Elektronen leiten; es steltt auf diese Weise einen Verstirkermechanismus dar und bewirkt eine Depolarization der Membran.
ONE of the unsolved problems of visual physiology is how only one quantum can start a nerve impulse in a retinal rod. Since a reaction involving possibly only one molecule of rhodopsin is amplified into a fully-fledged nerve impulse in the primary neurons, there must be some mechanism in the primary neurone which can act analogously to an amplifier. Two possible mechanisms by which amplification can occur (review, WALD, 1961a, 1961 b) are: (1) assuming the lamellae of the rod to be composed mostly of oriented molecules of rhodopsin (SJ~~STRAND, 1959; DENTON, 1958, 1959), the absorption of light by one of these molecules leaves a “hole” in the lamella so that local depolarization can occur by ion movement; (2) rhodopsin is a pro-enzyme, whereas one of the photoproducts is an enzyme (HECHT, 1918; JAHN, 1946, 1947) which might convert other pro-enzymes into enzymes which, in turn, start the nerve impulse. Either of these two mechanisms would provide 1 Supported by N.I.H. Grant No. 8611. 25
amplification, but neither has been proven to exist, and no details have been proposed concerning the nature of the hole. These and other less likely possibilities arc reviewed by CKESCITELLI (1960). This is a proposal of another mechanisn~ by wfhich amplifi~at~oI1 might occur in visual cells. Although this proposal is similar to the first of the two mechanisms mentioned above. it assumes that depolarization is caused by eIectron flow rather than ion movement. The location and mechanism of action proposed herein for the retinene portion of the rhodopsin molecule are, however, very different from those proposed by WALD (1961a, Fig. 4; 1961b). The possibility of electron flow along the carotenoid chain has been mentioned previously by DAKI‘NAM.(1948), but he did not relate this possibility to the isomeric changes to bc discussed below. It seems quite well established (WALD, 1961a) that one and possibly the only effect of light on any known visual system is to isomerize retinene. In the rhodopsin molecule, retinene occurs as II-cis- (neo-6) retinene, and the same statement is true of all visual pigments examined, including those from vertebrates, mollusks, and arthropods. Upon absorption of light the chromophore of rhodopsin is converted by stereoisomerization into the all-trans-retinene of lumirhodopsin. Lumirhodopsin is converted into metarhodopsin by a thermal rearrangement of the opsin portion of the molecule, and finally the metarhodopsin molecule is hydrolyzed to form all-trans-retinene and opsin. WULFF er al. (1958a), and WULFF et uE.(1958b) have described two fractions of photoproducts, one of which forms four more products. These discoveries are not contradictory to the conversion of isomers described by Wald and co-workers, but the two sets of data cannot be correlated because of the impossibility of identifying isomers during the llash pllotolysis techmique used by WCILFFef al. (1958a), or ofidentifying the fractions of Wulff and co-workers by the biochemical techniques used by Waid and co-workers. Possibly more than one new isomer is formed under the influence of light. One essential difference between the 1I-C& and the all-rvans structure is that the 1I-cls structure is hindered in its rotation about the single bond between carbon atoms 10 and 11 because of the cis- bonding at carbon 11, whereas the all-truns form is not so hindered. This difference permits the all-trans form to be a co-planar straight chain, whereas the I 1-cis cannot become co-planar. This results in a difference between the total maximum length of the retinene portion of the molecule of the two isomers of about 2 A. We may consider, therefore, that absorption of light increases the effective length of the retinene chain by about 2 A. In addition, the I I-cis form has very little resonance, while the all-trans form is highly resonant. These changes in the retinene molecule are discussed in detail by DARTNALL (1957). If the terminal keto group were free this lengthening could easily permit in a very simple manner the formation of an H-bond between the terminal keto group and some other compound, possibly with an imino group, a bonding which was prohibited previously by a deficiency in the length of the retinene chain. Or, as seems more possible, if the original bonding is at the keto end, the lengthening could permit bonding at the ring end of the chain, but the process, probably, could not be as simple as the formation of an H-bond. It seems as if this photophysical effect of light, which (1) makes an increase in length and a straightening of the chain possible, and which (2) greatly increases the degree of resonance in the chain, could permit electronic conduction through the chain and into-or away from---the other compound. There are at least several possibilities for the structure of this electron bridge, one of which is the arrangement of two molecules of retinene joined by protein or
A
PossibleMechanismfor the AmplifierEffect in the Retina
27
some simpler nitrogenous compound, similar to the structure of rhodopsin proposed by COLLINS and MORTON (1950; review, DARTNALL, 1957). The 9-&-isomer, which is unhindered and therefore resonant, can form a photosensitive pigment in combination with opsin, but this compound has never been extracted from any retina, This may indicate that, for visual stimulation to occur, light must cause an increase in conductivity (resonance) of the conjugated chain, as well as lengthening. If the high conductivity already exists, as in the 9-&s-isomer, an increase in length of the chain, although a prelude to bleaching, does not seem to permit the molecule to act as an amplifier in the membrane. If the opsin portion of the molecule is on a membrane surface, presumably in one of the lamellae of the rods, and the retinene portion is in the bimolecular lipoid portion of the membrane, the conduction of electrons could be through the membrane; that is, from one surface to the other. In this way light could convert the lipoid layer from the status of an electrical insulator to that of a semi-conductor. This increased electronic conductivity of the retinene portion of the molecule would exist as long as either lumirhodopsin or metarhodopsin exists, and also even after the molecule has been hydrolyzed. If, however, electronic conduction occurs through both the opsin and the retinene portions of the molecule, as seems most likely, the system would become non-conductive upon hydrolysis, if not sooner. This is in agreement with the statement of WALD (1961a) that “the first electrical response follows the stimulus by a small fraction of a second, [and] this leaves time for only the earliest steps in bleaching-the formation of the lumi- and at most the meta-pi~ent”. If the two sides of the membrane are at different electrical potentials, possibly so maintained by oxidation-reduction reactions (JAHN, 1962), the membrane can be considered to be analogous to a charged condenser with the charge stored in the reactants of the oxidation-reduction system. The only additional assumption needed is that electronic transfer can occur from reactants either on or near the membrane to opsin. This means that the opsin portion of the molecule acts either as an oxidation-reduction enzyme or is connected by resonance to such an enzyme. This proposal is essentially that a photophysical mechanism, by providing a conductor, may permit local depolarization of one of the lamellae of a rod by only one quantum. The degree of depolarization will depend on the electron pressure and the life span of the conductor. The question of how this local depolarization can spread to other parts of the lamella and eventually to the cell membrane to cause a nerve impulse in the primary neurone is still unanswered. One possibility for the mechanism of spreading is that conduction takes place not only perpendicularly through the lamella membranes, as proposed above, but also parallel to these membranes, either on the surface or through the lipoid layer of the lametla membrane to the cell membrane. The exact evaluation of this and of other possibilities must await a more detailed knowledge of the molecular structure of the membrane. Furthermore, the polarization of a lamellar membrane by oxidation-reduction systems could cause an unequal distribution of hydrogen ions by proton charge transfer (JAHN, 1962), and it seems possible that when the membrane is depolarized some of these charges might become redistributed by a rebound mechanism, thereby causing a shift in the hydrogen ion concentration of the two sides. The new part of this proposal is that the essential action of light, in isomerizing the retinene of the opsinretinene molecules, is to lengthen the retinene portion of the molecule and to increase its resonance, thereby making electronic conduction (DARTNALL, 1948) not
T. L. JhHh.
28
only
possible,
but
highly
probable
if a source of electrons is available from oxidation--
reduction reactions. REFERENCES COLLINS, F. D. and MORTON, R. A. (1950). Studies in rhodopsin. 3: Rhodopsin and transient orange. Biochem. J. 47, 18-24. CRESCITELLI, F. (1960). Physiology of vision. Ann. Rev. Ph_y.Gol.22, 525-578. DARTNALL, H. J. A. (1948). Indicator yellow and retinene. Nature, tend. 162, 122-123. DARTNALL. H. J. A. (1957). The Visual Pbments. Methuen & Co. Ltd.. London. 216 nn. DENTON, E: J. (1958). ‘Ligh; absorption by tie intact retina. Paper No. 5. Symposium Ni,k, Vi~suu/frohlems of Colour, 175-189. (Teddington, England.) DENTON, E. .I. (1959). The contributions of the oriented, photosensitive, and other molecules to the absorption of the whole retina. Proc. roy. Sot. 150B, 78-94. HECHT, S. (1918). The photic sensitivity of Ciona intestinalis. J. gen. Physiol. 1, 147-166. JAHN, T. L. (1946). Visual critical flicker frequency as a function of intensity. J. opt. Sot. Amer. 36,83-86. JAHN, T. L. (1947). Basic concepts in the interpretation of visual phenomena. Proc. lowa Acad. Sci. 54, 325-343.
JAHN, T. L. (1962). A theory of electronic conduction through membranes, and of active transport of ions. based on redox transmembrane potentials. J. theoret. Biol. 2, No. 2, 129-138. SJ~~STRAND,F. S. (1959). The ultrastructure of the retinal receptors of the vertebrate eye. Ergebn. Biol. 21, 128-260. WALD, G. (1961 a). The molecular organization of visual systems. Light nnd LiJ’k, W. D. MCELROY and B. GLASS (Editors). 724-749. Johns Hopkins Press, Baltimore. U.S.A. 924 uix WALD, C. (ldbl b). C&era1 discussion of retinal structure in relation to the visd process. The Structure of the Eye, 101-t 15. Academic Press, New York. WULFF, V. J., ADAMS, R. G., LINSCHZTZ,H. and ABRAHAMSON,E. W. (1958a). Effect of flash illumination on rhodopsin in solution. Ann. N. Y. .&ark Sci. 74, 281-290. WULFF, V. J., ADAMS, R. G., LINSCHITZ, H. and KENNEDY, D. (t958b). The behavior of nash-illuminated rhodopsin in solution. A.M.A. Arrh. Q~ht~~a~.60, 695.-701.
Pergamon Press 1963. Printed in Great Britain.
A POSSIBLE
MECHANIST EFFECT
FOR
IN THE
THE
AMPLIFIER
RETINA’
THEODORE LOUIS JAHN Department of Zoology, University of Caiifornia, Los Angeles (Received 31 Augusr 1962)
transforms the 1I-cis-retinene of the rhodopsin molecule into all-transretinene, thereby making the molecule longer and highly resonant. It is proposed that these changes permit electron flow through the lamellar membranes, with the electrons provided by and received by oxidation-reduction enzymes, one on each side of a membrane. This conductive state of retinene occurs only in the lumi- and metarhodopsins. While conductive, a molecule of retinene, under electron pressure from oxidation-reduction enzymes, might conduct many electrons, thereby providing an amplifier mechanism, and causing depolariz&on of the membrane. Abstract-Light
R&urn&La lumi&re transforme le 11-cis&in&ne de la mol&ule de rhodopsine en transr&int?ne, ce qui rend la mol&ule plus longue et fortement r&onante. On propose que ces changements permettent le passage des tlectrons it travers les membranes lamellaires; il s’agirait des Clectrons lib&s ou cap& par les enzymes d’oxydo-tiduction, un de chaque c&t! de la membrane. Cet &tat de conduction du r&intne n’existerait que pour la lumi- et m&arhodopsine. Dam cet &at conducteur, et sous la pression des Bectrons provenant des enzymes d’oxyd*r&duction, la molt%ule de r&in&e pourrait conduire beaucoup d’&ectrons, se comportant ainsi comme un m&canisme ampiificateur et d&polar&ant la membrane. Z~rne~~~g-Licbt fiihrt das Cis-Retinen des Rhodopsinmolek~ls in Trans-Retinen ijber. Das MoIekSl wird dabei linger und in starkem Ausmasse resonanz~hig. Es wird angenom~, dass diese V~~nde~ngen einen Eiektronenstrom durch die lamellena~igen Membranen zulassen, wobei die E~ektronen van den ~yda~io~s-Reduktions-Enzymen abgegeben und a~genommen werden, die sich auf jeder Seite einer Membran befinden. Dieser leitende Z&and des Retinens tritt nur bei Lumi- und Metarhodopsjnen auf. Ein Retinenmojek~l kann so, wghrend es leitend ist, unter dem Elektronendruck der OxydationsReduktions-Enzyme viele Elektronen leiten; es steltt auf diese Weise einen Verstirkermechanismus dar und bewirkt eine Depolarization der Membran.
ONE of the unsolved problems of visual physiology is how only one quantum can start a nerve impulse in a retinal rod. Since a reaction involving possibly only one molecule of rhodopsin is amplified into a fully-fledged nerve impulse in the primary neurons, there must be some mechanism in the primary neurone which can act analogously to an amplifier. Two possible mechanisms by which amplification can occur (review, WALD, 1961a, 1961 b) are: (1) assuming the lamellae of the rod to be composed mostly of oriented molecules of rhodopsin (SJ~~STRAND, 1959; DENTON, 1958, 1959), the absorption of light by one of these molecules leaves a “hole” in the lamella so that local depolarization can occur by ion movement; (2) rhodopsin is a pro-enzyme, whereas one of the photoproducts is an enzyme (HECHT, 1918; JAHN, 1946, 1947) which might convert other pro-enzymes into enzymes which, in turn, start the nerve impulse. Either of these two mechanisms would provide 1 Supported by N.I.H. Grant No. 8611. 25
amplification, but neither has been proven to exist, and no details have been proposed concerning the nature of the hole. These and other less likely possibilities arc reviewed by CKESCITELLI (1960). This is a proposal of another mechanisn~ by wfhich amplifi~at~oI1 might occur in visual cells. Although this proposal is similar to the first of the two mechanisms mentioned above. it assumes that depolarization is caused by eIectron flow rather than ion movement. The location and mechanism of action proposed herein for the retinene portion of the rhodopsin molecule are, however, very different from those proposed by WALD (1961a, Fig. 4; 1961b). The possibility of electron flow along the carotenoid chain has been mentioned previously by DAKI‘NAM.(1948), but he did not relate this possibility to the isomeric changes to bc discussed below. It seems quite well established (WALD, 1961a) that one and possibly the only effect of light on any known visual system is to isomerize retinene. In the rhodopsin molecule, retinene occurs as II-cis- (neo-6) retinene, and the same statement is true of all visual pigments examined, including those from vertebrates, mollusks, and arthropods. Upon absorption of light the chromophore of rhodopsin is converted by stereoisomerization into the all-trans-retinene of lumirhodopsin. Lumirhodopsin is converted into metarhodopsin by a thermal rearrangement of the opsin portion of the molecule, and finally the metarhodopsin molecule is hydrolyzed to form all-trans-retinene and opsin. WULFF er al. (1958a), and WULFF et uE.(1958b) have described two fractions of photoproducts, one of which forms four more products. These discoveries are not contradictory to the conversion of isomers described by Wald and co-workers, but the two sets of data cannot be correlated because of the impossibility of identifying isomers during the llash pllotolysis techmique used by WCILFFef al. (1958a), or ofidentifying the fractions of Wulff and co-workers by the biochemical techniques used by Waid and co-workers. Possibly more than one new isomer is formed under the influence of light. One essential difference between the 1I-C& and the all-rvans structure is that the 1I-cls structure is hindered in its rotation about the single bond between carbon atoms 10 and 11 because of the cis- bonding at carbon 11, whereas the all-truns form is not so hindered. This difference permits the all-trans form to be a co-planar straight chain, whereas the I 1-cis cannot become co-planar. This results in a difference between the total maximum length of the retinene portion of the molecule of the two isomers of about 2 A. We may consider, therefore, that absorption of light increases the effective length of the retinene chain by about 2 A. In addition, the I I-cis form has very little resonance, while the all-trans form is highly resonant. These changes in the retinene molecule are discussed in detail by DARTNALL (1957). If the terminal keto group were free this lengthening could easily permit in a very simple manner the formation of an H-bond between the terminal keto group and some other compound, possibly with an imino group, a bonding which was prohibited previously by a deficiency in the length of the retinene chain. Or, as seems more possible, if the original bonding is at the keto end, the lengthening could permit bonding at the ring end of the chain, but the process, probably, could not be as simple as the formation of an H-bond. It seems as if this photophysical effect of light, which (1) makes an increase in length and a straightening of the chain possible, and which (2) greatly increases the degree of resonance in the chain, could permit electronic conduction through the chain and into-or away from---the other compound. There are at least several possibilities for the structure of this electron bridge, one of which is the arrangement of two molecules of retinene joined by protein or
A
PossibleMechanismfor the AmplifierEffect in the Retina
27
some simpler nitrogenous compound, similar to the structure of rhodopsin proposed by COLLINS and MORTON (1950; review, DARTNALL, 1957). The 9-&-isomer, which is unhindered and therefore resonant, can form a photosensitive pigment in combination with opsin, but this compound has never been extracted from any retina, This may indicate that, for visual stimulation to occur, light must cause an increase in conductivity (resonance) of the conjugated chain, as well as lengthening. If the high conductivity already exists, as in the 9-&s-isomer, an increase in length of the chain, although a prelude to bleaching, does not seem to permit the molecule to act as an amplifier in the membrane. If the opsin portion of the molecule is on a membrane surface, presumably in one of the lamellae of the rods, and the retinene portion is in the bimolecular lipoid portion of the membrane, the conduction of electrons could be through the membrane; that is, from one surface to the other. In this way light could convert the lipoid layer from the status of an electrical insulator to that of a semi-conductor. This increased electronic conductivity of the retinene portion of the molecule would exist as long as either lumirhodopsin or metarhodopsin exists, and also even after the molecule has been hydrolyzed. If, however, electronic conduction occurs through both the opsin and the retinene portions of the molecule, as seems most likely, the system would become non-conductive upon hydrolysis, if not sooner. This is in agreement with the statement of WALD (1961a) that “the first electrical response follows the stimulus by a small fraction of a second, [and] this leaves time for only the earliest steps in bleaching-the formation of the lumi- and at most the meta-pi~ent”. If the two sides of the membrane are at different electrical potentials, possibly so maintained by oxidation-reduction reactions (JAHN, 1962), the membrane can be considered to be analogous to a charged condenser with the charge stored in the reactants of the oxidation-reduction system. The only additional assumption needed is that electronic transfer can occur from reactants either on or near the membrane to opsin. This means that the opsin portion of the molecule acts either as an oxidation-reduction enzyme or is connected by resonance to such an enzyme. This proposal is essentially that a photophysical mechanism, by providing a conductor, may permit local depolarization of one of the lamellae of a rod by only one quantum. The degree of depolarization will depend on the electron pressure and the life span of the conductor. The question of how this local depolarization can spread to other parts of the lamella and eventually to the cell membrane to cause a nerve impulse in the primary neurone is still unanswered. One possibility for the mechanism of spreading is that conduction takes place not only perpendicularly through the lamella membranes, as proposed above, but also parallel to these membranes, either on the surface or through the lipoid layer of the lametla membrane to the cell membrane. The exact evaluation of this and of other possibilities must await a more detailed knowledge of the molecular structure of the membrane. Furthermore, the polarization of a lamellar membrane by oxidation-reduction systems could cause an unequal distribution of hydrogen ions by proton charge transfer (JAHN, 1962), and it seems possible that when the membrane is depolarized some of these charges might become redistributed by a rebound mechanism, thereby causing a shift in the hydrogen ion concentration of the two sides. The new part of this proposal is that the essential action of light, in isomerizing the retinene of the opsinretinene molecules, is to lengthen the retinene portion of the molecule and to increase its resonance, thereby making electronic conduction (DARTNALL, 1948) not
T. L. JhHh.
28
only
possible,
but
highly
probable
if a source of electrons is available from oxidation--
reduction reactions. REFERENCES COLLINS, F. D. and MORTON, R. A. (1950). Studies in rhodopsin. 3: Rhodopsin and transient orange. Biochem. J. 47, 18-24. CRESCITELLI, F. (1960). Physiology of vision. Ann. Rev. Ph_y.Gol.22, 525-578. DARTNALL, H. J. A. (1948). Indicator yellow and retinene. Nature, tend. 162, 122-123. DARTNALL. H. J. A. (1957). The Visual Pbments. Methuen & Co. Ltd.. London. 216 nn. DENTON, E: J. (1958). ‘Ligh; absorption by tie intact retina. Paper No. 5. Symposium Ni,k, Vi~suu/frohlems of Colour, 175-189. (Teddington, England.) DENTON, E. .I. (1959). The contributions of the oriented, photosensitive, and other molecules to the absorption of the whole retina. Proc. roy. Sot. 150B, 78-94. HECHT, S. (1918). The photic sensitivity of Ciona intestinalis. J. gen. Physiol. 1, 147-166. JAHN, T. L. (1946). Visual critical flicker frequency as a function of intensity. J. opt. Sot. Amer. 36,83-86. JAHN, T. L. (1947). Basic concepts in the interpretation of visual phenomena. Proc. lowa Acad. Sci. 54, 325-343.
JAHN, T. L. (1962). A theory of electronic conduction through membranes, and of active transport of ions. based on redox transmembrane potentials. J. theoret. Biol. 2, No. 2, 129-138. SJ~~STRAND,F. S. (1959). The ultrastructure of the retinal receptors of the vertebrate eye. Ergebn. Biol. 21, 128-260. WALD, G. (1961 a). The molecular organization of visual systems. Light nnd LiJ’k, W. D. MCELROY and B. GLASS (Editors). 724-749. Johns Hopkins Press, Baltimore. U.S.A. 924 uix WALD, C. (ldbl b). C&era1 discussion of retinal structure in relation to the visd process. The Structure of the Eye, 101-t 15. Academic Press, New York. WULFF, V. J., ADAMS, R. G., LINSCHZTZ,H. and ABRAHAMSON,E. W. (1958a). Effect of flash illumination on rhodopsin in solution. Ann. N. Y. .&ark Sci. 74, 281-290. WULFF, V. J., ADAMS, R. G., LINSCHITZ, H. and KENNEDY, D. (t958b). The behavior of nash-illuminated rhodopsin in solution. A.M.A. Arrh. Q~ht~~a~.60, 695.-701.