Scientific Retina International‘s

Scientific Newsletter


The Visual Cascade

Recent update from: 12.12.1999


This page introduces you to the visual cascade.
You may click on a protein symbol on the enlarged map and you will receive a description of the chosen step. The description includes links to further pages providing information on that protein.

For a high quality copy of the schematics you may inquire at the pagemaster’s office .
The
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Rhodopsin | Transducin | cGMP Phosphodiesterase | Cyclic Nucleotide-gated Cation Channel



The Visual Cascade - From Photon to Electrical Response

Vision is a process triggered by the uptake of photons via a photosensitive protein, conversion, and amplification of the trigger into an electrical response via the visual cascade, and the subsequent generation of electrical nerve impulses that are sent to the brain.
Most of the data, published on mammalian phototransduction, refer to bovine rod cells or other animal systems due to better accessibility of specimen. Therefore, data reported below and the related pages of this site cannot directly be transfered to the human biochemistry of vision. We will refer to known differences to the human system if necessary.

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The The Photopigments

There are four human light perceiving proteins (photopigments) sharing homologies of about 41% to each other. Only Rhodopsin (RHO) with an absorption maximum at 495 nm (21) is present in rod photoreceptors. In cone photoreceptors photopigments for light absorption at 420 nm (blue cone pigment (BCP)), 530 nm (green cone pigment (GCP)) or 560 nm (red cone pigment (RCP)) respectively are used (22) .
All photopigments resemble the 7 alpha-helix structure of G-protein coupled receptors. They are inserted into the plasma membrane of rods and cones, as well as into the disc membrane of rods. Due to the fact that the plasma membrane of the outer segment of rod photoreceptors stands for only 4% of the whole membrane it is neglectable concerning the photopigments inserted into it.
To a Lysine residue (RHO: Lys 296) in the seventh transmembrane domain of the photopigment an 11-cis retinaldehyde is attached via a Schiff bond. 11 cis-retinaldehyde, the chromophore, changes its conformation to all-trans retinaldehyde on the absorption of a photon and therefore changes the conformation of the holoprotein, herein referred to as metarhodopsin II (24). For metarhodopsin II the binding sites for regulators like Rhodopsin Kinase (RK) or Arrestin (S-antigen (SAG) or 48-kDa protein), and for the alpha-subunit of Transducin (GNAT), which connects the trigger with the amplification cascade, are accessible to these proteins.

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Transducin The G-protein Transducin

After light perception Transducin (the adapter) binds to the excited photopigments to pick up the trigger (retinaldehyde isomerisation) and proceed it onto the amplification mechanism of the visual cascade. Excited photopigments bind several Transducin molecules one after another. This first step leads to a 102-103 times amplification of the trigger
(6).

Transducin is a heterotrimer consisting of 3 different subunits: the catalytic alpha-subunit (GNAT) and the regulatory beta (GNB) and gamma-subunits (GNG), that form an active heterodimer. The proteins of the Transducin complex belong to a family of GTP-binding proteins (G proteins) that connect different receptors with their second messenger pathway via their alpha-subunit. The variety of receptors and effectors served by the heterotrimeric G-proteins has evolutionary created a multiplicity of alpha-subunit isoforms for the different receptors.
Dealing with human phototransduction we have to focus on two alpha-subunits (13) (20) that are abundant to the different types of photoreceptor cells (GNAT1 in rods, GNAT2 in cones). The same holds for the beta-subunits (12) (19). Two of the three described Transducin beta-subunits (GNB) are found in the retina (18) (17) where GNB1 is found specifically in rods and GNB3 in cones. Today a set of 8 gamma-subunits (GNG) is biochemically described , from different kinds of tissues. GNG1 and GNG3 (5) (16) are reported to be specific for rod outer segments (ROS) and brain respectively and GNG8 is found in cones (23) while the remaining 5 gamma-subunits are less abundant to a specific tissue.

The interaction of the Transducin complex with the different photopigments is dependent on GNAT and GNG (16) while the beta-subunit is necessary for forming the heterotrimeric complex that is able to bind to the receptor protein. The Transducin complex is loosely attached to the disc membranes of rods and the plasma membranes of cones. Bound to GNAT , GNB-GNG dimer stays attached to the membrane, while after dissociation of GNAT it can detach from the membrane. GNAT binds phosphatidylethanolamine and myristic acid to enhance the membrane attachment. The attachment to the GNB-GNG dimer, while GDP is bound, form the GNAT-GNB-GNG trimer. On interaction with the excited photopigments, GNAT exchanges bound GDP for GTP. This leads to detachment from the photopigment and from GNB-GNG dimer. Subsequently GNAT slides along the membrane to interact with the gamma-subunit dimer of cGMP Phosphodiesterase (PDEG 2 ). GNAT hydrolyses GTP to GDP and inorganic phosphate on binding to PDEG 2 .GTP hydrolysis is enhanced by RGS9 which acts as a guanylase activating protein. (14) .
After GTP hydrolysis GNAT is released from PDEG 2 and reassociates with GNB-GNG to start the cycle again on binding to Rhodopsin (RHO) (24)

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The The Phosphodiesterases

The effector of the visual cascade is cGMP Phosphodiesterase (PDE) , a heterotetrameric protein attached to the disc membrane. PDE hydrolyses 5'3'cGMP to 5'GMP PDE alpha- (PDEA) and beta-subunits (PDEB) are catalytic subunits, that stay together as a dimeric complex in rods while in cones there is a PDEA'-dimer. PDEA, PDEA', and PDEB share great homologies (9) (26) . The inhibitory subunit is a dimer of two gamma-subunits (PDEG 2 ) that binds to activated GNAT (24) . The activity of PDE is regulated threefold by the attachment of GNAT to PDEG 2 :
(10) (i) in darkness, there is a low basic cGMP turnover by PDE without binding of GNAT , the cGMP concentration in ROS is high, and the non catalytic cGMP binding sites of PDEA-PDEB are occupied;
(ii) on attaching GNAT to PDEG 2 , PDEG 2 keeps bound to the PDE complex , activating the cGMP hydrolysis;
(iii) after illumination for some time, the cGMP concentrations in ROS become reduced due to cGMP-PDE activity. PDEA-PDEB non catalytic cGMP-binding sites are set free from cGMP and PDEG 2 can dissociates into the cytosol on binding of GNAT . The aim of this mechanism is to adjust the PDE affinity for the substrate cGMP. On high cGMP concentration, PDE adjusts the steady state cGMP level of the photoreceptor outer segment with reduced cGMP affinity due to PDEG blocks. On illumination its cGMP-affinity is enhanced and the turn over rates increase significantly with reduction of the cGMP concentrations. There are two regions for PDEG 2 to get into contact with PDEA-PDEB : a basic region at residues 24-45 of PDEG , that also attaches to GNAT (residues 245 - 270), and an inhibitory region at residues 88 - 87 of PDEG , that shows lower affinity to GNAT (residues 306 - 310). (3) (2) Activation of PDE is the last step of amplification in the visual cascade and produces a 105 fold amplification of the primary trigger due to PDE activity while GNAT stays attached to PDEG2 (6) .

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The Creation of the electrical impulse- The Cyclic Nucleotide-gated Cation Channel

The last step in phototransduction is the creation of the nerve impulse. This is mediated by the Cyclic Nucleotide-gated Cation Channel (CNCG).
In the dark the channel is co-operatively kept open on binding of 4 cGMP molecules with its beta-subunits (4)
This causes an exchange of cations (Ca2+, Na+, K+) between the cytoplasm and the surrounding interphotoreceptor space. To keep the ion gradients active the cations are actively pumped across the plasmamembrane by Na+/Ca2+/K+-Exchanger. (25)
In the light cGMP is hydrolysed to 5'GMP by PDEA-PDEB . With decreased cGMP concentration cGMP is removed from CNCG2-CNCG3 and the channel is closed. This blocks the flow of Ca 2+ and Na + inside the ROS. In darkness the inward flow of charges (Na + , K + , Ca 2+ ) is equal to the outward flow. This is obtained by a Na + /K + /Ca 2+ -exchanger in the ROS membrane. At illumination the Na + /K + /Ca 2+ -exchanger is still active but the inward Ca 2+ and Na + flow through the CNCG is blocked and the plasma membrane is hyperpolarised because the charge flow rates have become unequal. This hyperpolarisation leads to the nerve impulse that is sent to the brain (27) . CNCG is also liable to high calmodulin-Ca 2+ , that leads to closure of the channel to reduce the Ca 2+ influx (15) (7) . The CNCG is a non selective channel for alkali cations in the plasmamembrane. The channel is made up of 3 different subunits. CNCG1 is the channelling protein, while CNCG2 and CNCG3 have regulatory function (11) (8) (1). As a homotetramer CNCG1 forms a channel that supports an exchange of monovalent ions without discrimination and a Ca 2+ inward current in the dark. The beta-subunits have been characterised in mice (8) (1) . The CNCG2-CNCG3 subunits are covalently linked to each other. They are co-purified as a 240 kDa dimer that includes a 63 kDa protein in mouse retina, which resembles the electrophoretic properties of CNCG2 . As expected on its introduction in 1994, the glutamic acid rich protein (GAR-1) was found to be the CNCG3 subunit of the CNCG .

Every step of the primary pathway of phototransduction has been found to be involved in retinal degenerations. For further data see the mutation database and the protein pages hyperlinked above.


References
  1. Ardell,M.D., Makhija,A.K., Viegas-Pequignot,E., Miniou,P., and Pittler,S.J. Molecular analysis of the human GAR-1 locus. 1995; Invest.Ophthalmol.Vis.Sci. 36: S774
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  2. Artemyev,N.O., Mills,J.S., Thornburg,K.R., Knapp,D.R., Schey,K.L., and Hamm,H.E. A site on transducin alpha-subunit of interaction with the polycationic region of cGMP phosphodiesterase inhibitory subunit. 1993; J.Biol.Chem. 268: 23611-23615.
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  3. Artemyev,N.O., Rarick,H.M., Mills,J.S., Skiba,N.P., and Hamm,H.E. Sites of interaction between rod G-protein alpha-subunit and cGMP-phosphodiesterase gamma-subunit. Implications for the phosphodiesterase activation mechanism. 1992; J.Biol.Chem. 267: 25067-25072.
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  4. Brown,R.L., Gramling,R., Bert,R.J., and Karpen,J.W. Identification by photoaffinity labeling of peptide regions within retinal rod cGMP-activated channel subunits involved in cGMP binding. 1994; Invest.Ophthalmol.Vis.Sci. 35 (Suppl.): 1473
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  5. Cali,J.J., Balcueva,E.A., Rybalkin,I., and Robishaw,J.D. Selective tissue distribution of G protein gamma subunits, including a new form of the gamma subunits identified by cDNA cloning. 1992; J.Biol.Chem. 267: 24023-24027.
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  6. Chabre,M. and Deterre,P. Molecular mechanism of visual transduction. 1989; Eur.J.Biochem. 179: 255-266.
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  7. Chen,T.Y., Illing,M., Molday,L.L., Hsu,Y.T., Yau,K.W., and Molday,R.S. Subunit 2 (or beta) of retinal rod cGMP-gated cation channel is a component of the 240-kDa channel-associated protein and mediates Ca(2+)-calmodulin modulation. 1994; Proc.Natl.Acad.Sci.U.S.A. 91: 11757-11761.
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  8. Chen,T.Y., Peng,Y.W., Dhallan,R.S., Ahamed,B., Reed,R.R., and Yau,K.W. A new subunit of the cyclic nucleotide-gated cation channel in retinal rods. 1993; Nature. 362: 764-767.
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  9. Collins,C., Hutchinson,G., Kowbel,D., Riess,O., Weber,B., and Hayden,M.R. The human beta-subunit of rod photoreceptor cGMP phosphodiesterase: Complete retinal cDNA sequence and evidence for expression in brain. 1992; Genomics. 13: 698-704.
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  10. Cunnick,J., Twamley,C., Udovichenko,I., Gonzalez,K., and Takemoto,D.J. Identification of a binding site on retinal transducin alpha for the phosphodiesterase inhibitory gamma subunit. 1994; Biochemical.Journal. 297: 87-91.
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  11. Dhallan,R.S., Macke,J.P., Eddy,R.L., Shows,T.B., Reed,R.R., Yau,K.W., and Nathans,J. Human rod photoreceptor cGMP-gated channel: amino acid sequence, gene structure, and functional expression. 1992; Journal.of.Neuroscience. 12: 3248-3256.
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  12. Fong,H.K., Amatruda,T.T., Birren,B.W., and Simon,M.I. Distinct forms of the beta subunit of GTP-binding regulatory proteins identified by molecular cloning. 1987; Proc.Natl.Acad.Sci.U.S.A. 84: 3792-3796.
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  13. Fong,S.L. Characterization of the human rod transducin alpha-subunit gene. 1992; Nucleic.Acids.Res. 20: 2865-2870.
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  14. He,L., Swaroop,A., and Fox,D.A. Spatiotemporal Pattern Of NRL In The Developing Rat Retina. 1998; Invest.Ophthalmol.Vis.Sci. 39: S197
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  15. Hsu,Y.T. and Molday,R.S. Interaction of calmodulin with the cyclic GMP-gated channel of rod photoreceptor cells. Modulation of activity, affinity purification, and localization. 1994; J.Biol.Chem. 269: 29765-29770.
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  16. Kisselev,O. and Gautam,N. Specific interaction with rhodopsin is dependent on the gamma subunit type in a G protein. 1993; J.Biol.Chem. 268: 24519-24522.
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  17. Lee,R.H., Lieberman,B.S., Yamane,H.K., Bok,D., and Fung,B.K. A third form of the G protein beta subunit. 1. Immunochemical identification and localization to cone photoreceptors. 1992; J.Biol.Chem. 267: 24776-24781.
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  18. Lee,R.H., Ting,T.D., Lieberman,B.S., Tobias,D.E., Lolley,R.N., and Ho,Y.K. Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin. 1992; J.Biol.Chem. 267: 25104-25112.
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  19. Levine,M.A., Smallwood,P.M., Moen,P.T.J., Helman,L.J., and Ahn,T.G. Molecular cloning of beta 3 subunit, a third form of the G protein beta-subunit polypeptide. 1990; Proc.Natl.Acad.Sci.U.S.A. 87: 2329-2333.
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  20. Morris,T.A. and Fong,S.L. Characterization of the gene encoding human cone transducin alpha-subunit (GNAT2). 1993; Genomics. 17: 442-448.
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  21. Nathans,J. and Hogness,D.S. Isolation and nucleotide sequence of the gene encoding human rhodopsin. 1984; Proc.Natl.Acad.Sci.U.S.A. 81: 4851-4855.
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  22. Nathans,J., Thomas,D., and Hogness,D.S. Molecular genetics of human color vision: the genes encoding blue, green, and red pigments. 1986; Science. 232: 193-202.
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  23. Ong,O.C., Yamane,H.K., Phan,K.B., Fong,H.K., Bok,D., Lee,R.H., and Fung,B.K. Molecular cloning and characterization of the G protein gamma subunit of cone photoreceptors. 1995; J.Biol.Chem. 270: 8495-8500.
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  24. Stryer,L. Visual excitation and recovery Visual excitation and recovery. 1991; J.Biol.Chem. 266: 10711-10714.
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  25. Tucker,J.E., Winkfein,R.J., Cooper,C.B., and Schnetkamp,P.P. cDNA cloning of the human retinal rod Na-Ca + K exchanger: comparison with a revised bovine sequence. 1998; Invest.Ophthalmol.Vis.Sci. 39: 435-440.
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  26. Viczian,A.S., Piriev,N.I., and Farber,D.B. Isolation of a cDNA encoding the alpha’ subunit of human cone cGMP- phosphodiesterase. 1994; Invest.Ophthalmol.Vis.Sci. 35: 1264
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  27. Yau,K.W. Phototransduction mechanism in retinal rods and cones. The Friedenwald Lecture. 1994; Invest.Ophthalmol.Vis.Sci. 35: 9-32.
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This site is maintained and edited by
Dr. rer. medic. Markus Preising, Dipl.Biol.
Molecular Genetics Laboratory
Department of Paediatric Ophthalmology, Strabismology and Ophthalmogenetics
University of Regensburg
Head: Prof. Dr. med. Birgit Lorenz