The Photopigments
Recent update from: 12.12.99
This page introduces you to the visual cascade.
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description of the chosen step. The description includes links to further pages
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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.
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.
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)
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)
.
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.
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
Goto Top
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.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
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
Goto Top
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.
Goto Top
Link to PudMed
6. Chabre, M. and Deterre, P. Molecular
mechanism of visual transduction. 1989; Eur.J.Biochem. 179: 255 - 266.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
13. Fong, S.L. Characterization of the human
rod transducin alpha-subunit gene. 1992; Nucleic.Acids.Res. 20: 2865 - 2870.
Goto Top
Link to PudMed
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
Goto Top
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.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
20. Morris, T.A. and Fong, S.L.
Characterization of the gene encoding human cone transducin alpha-subunit (GNAT2).
1993; Genomics. 17: 442 - 448.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
24. Stryer, L. Visual excitation and recovery
Visual excitation and recovery. 1991; J.Biol.Chem. 266: 10711 - 10714.
Goto Top
Link to PudMed
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.
Goto Top
Link to PudMed
26. Viczian, A.S., Piriev, N.I., and Farber,
D.B. Isolation of a cDNA encoding the ?'subunit of human cone
cGMP-phosphodiesterase. 1994; Invest.Ophthalmol.Vis.Sci. 35: 1264
Goto Top
27. Yau, K.W. Phototransduction mechanism in
retinal rods and cones. The Friedenwald Lecture. 1994; Invest.Ophthalmol.Vis.Sci.
35: 9 - 32.
Goto Top
Link to PudMed
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The G-protein Transducin
The
Phosphodiesterases