Implantación del AMFE

The outer segment of photoreceptor cells contains several hundred thin membrane

plates (lamellae). In rods, the lamellae are free floating disks, whereas in cones they consist

o f one continuous folded membrane. Embedded in the lamellae membrane are the visual

pigment molecules, which are the most abundant protein within the outer segment,

accounting for approximately 80% of the membrane proteins present (Nathans; 1987). The

absorption of photons by these light-sensitive pigments triggers a conformational change

that eventually modifies a neural output from the photoreceptor cell (section 1.6). The

biosynthetic machineiy for the production o f photopigments and other components of

visual transduction is situated within the inner segment o f photoreceptor cells. The actual

mechanism of transport of these components from the inner segment to the outer segment is

Figure 1.5: Rod and cone photoreceptor cells

(adapted from All and Kline 1985).

O U T E R S E G M E N T II C O N N E C T I N G C I L I U M I N N E R S E G M E N T N U C L E U S R E C E P T O R T E R M I N A L Rod Cone -31 -

The visual pigments belong to a large superfamily of structurally similar integral

membrane proteins, the G-protein-linked receptors (Baldwin 1993; Schertler et a i, 1993;

Alkorta and Du, 1994). They are characterised by seven hydrophobic a-helical membrane-

spanning segments (helices I-VII) that are linked by extramembrane hydrophilic loops

(Khorana 1992; Trumpp-Kallmeyer et a i, 1992). The amino-terminal (N-terminal) and the

carboxy-terminal (C-terminal) of the protein lie within the extracellular and cytoplasmic

regions of the cell membrane, respectively (figure 1.6).

Figure 1.6: Transmembrane structure o f human opsins

Tw o dim ensional representation o f the opsin protein. HOOC and Ac-N denote carboxy and am ino terminal ends respectively. The differences indicated are between the human L and M opsins (section 1.9).

( ylopWwn

Kxt«mal

Mirfiice _{# Retinal binding site}

• Non-conserved substitutions O Conserved substitutions

The human retina has four photopigments, one rod and three cone photopigments, all of

similar construction. Each pigment molecule consists of two parts: a large protein moiety,

the opsin, which is connected by a covalent Schiff-base linkage with a lysine residue in the

seventh helix of the opsin molecule, to a chromophore. The chromophore is usually the 11-

cis isomer of vitamin A aldehyde (ll-cw -retinal, figure 1.7.1) which forms a rhodopsin

molecule with opsin. Retinal is a long-chain molecule that can exist in two forms or

isomers: a straight chain form called all-tran^-retinal, and a bent form, ll-ci5-retinal which

is the only form that can bind to the opsin. The retinal is the portion of the photopigment

first affected by light absorption and it is considered to be the active part of the opsin-retinal

complex (section 1.6). The seven membrane helices (helices I to VII) form a pocket within

which the chromophore is situated (figure 1.7.2). In human photopigments, only the opsin

differs from one pigment to another. The differences among photopigment opsins result in

differences in the wavelengths of light that these photopigments preferentially absorb. Each

pigment absorbs maximally at a different wavelength, termed its lambda max (>Snax)» which

can be used to characterise the pigment (figure 1.8).

1,5.1 Rod Opsin

Following the publication of the amino acid sequence of bovine rod opsin

(Ovchinnikov, 1982; Hargrave et al, 1983), the genes encoding bovine (Nathans and

Hogness, 1983) and human (Nathans and Hogness, 1984) rod opsins were sequenced. The

human rod opsin protein is 348 amino acids in length, shares 94% identity with bovine rod

opsin and has a o f 496nm. Human rod opsin forms the seven membrane spanning

structure as previously described (section 1.5) and the structural and functional properties

of rod opsin have been reviewed extensively (Nathans, 1992; Maden, 1995). More recently

the crystal structure of bovine rod rhodopsin has been revealed (Palczewski et al., 2000).

Figure 1.7: Retinal and opsin palisade. 1) Ih e absorption o f light causes photoisom erization of ll-c/5-retinal ( la ) to all-rran5-retinal (Ih ) and 2) the ‘palisade’ arrangem ent thought to depict the three dim ensional conform ation of the functional opsin molecule. HOOC and Ac-N denote the carhoxy and am ino terminal ends respectively. Amino acid residues identified are residues thought to he important for the spectral tuning o f the visual pigm ent (section 1.5.2).

la) H3C CM l b ) H3C CH3 ÇH3 13 nak 115 c 1 1- c i s - R c t i n a l _{A\\-Trans-Retma\} 2) Ac-N lixiniccllulur MOOC A la-285 Intracellular C O O H Ala -180 N-A c .Scr-233 l^e-277-

too 1 80 RE LA TIV E ABSO RBA N CE (to -4 40 ^ 20 - 400 500 600 W A V ELEN G TH (m n) 700

Figure 1.8: The spectral sensitivities o f the three types o f normal human cone photoreceptors. The blue, green and red curves represent the S, M and L photopigm ents respectively. Adapted from K.Dulai, 1996.

Functional and sequence analysis of rod opsins from different species has revealed

certain amino acids that are conserved in all opsins, indicating that they are essential for the

proper activity of these proteins. The protonated Schiff base at Lys-296, which sits within

helix VII, binds the chromophore 11-cw-retinal thus enabling absorption of visible light by

rod opsin. To stabilise the positive charge created by the Schiff base, a second site was

predicted. Site directed mutagenesis experiments identified the counterion as Glu-113

(Sakmar et al., 1989; Zhukovsky and Oprian, 1989). In terms of the tertiary structure, these

two residues face the retinal binding pocket in the centre of the ring of transmembrane

helices (Baldwin, 1993). Two cysteine residues, Cys-IIO and Cys-187, situated within

extracellular loops I and II respectively, form a disulphide bond (Karnik and Khorana,

1990) and, together, are essential for the proper structural formation o f rod opsin (Kamik et

al., 1988). The cytoplasmic loop II, that connects helices III and IV, and cytoplasmic loop

III, which connects helices V and VI, and specific regions within the C-terminal region of

rod opsin are essential for the activation of the G-protein, transducin (Konig et al., 1989).

7.5.2 Cone Opsins

Human cone photoreceptor cells contain one of three cone opsins that absorb light

maximally in different regions of the spectrum (figure 1.8). Structural differences between

the proteins govern the spectral properties of these pigments. Indeed, the cone

photopigments can be classed according to their X-max (Bowmaker et al., 1980;

Bowmaker, 1984). Short wavelength-sensitive (S) cones have a photopigment which

absorbs maximally at 419nm, while middle wavelength-sensitive (M) cones and long

wavelength-sensitive (L) cones have a of 531nm and 558nm respectively (Oprian et

al., 1991; Merbs and Nathans, 1992). While cone pigments which have a X^ax of 419nm,

531nm and 558nm may be referred to as blue, green and red respectively, this terminology

can be misleading since each of the cone opsins does not necessarily absorb maximally

within the blue, green and red region of the visible spectrum. For instance, the X^ax for the

human red cone pigment actually corresponds to the green-yellow region of the visible

spectrum, while the for the human blue cone pigment corresponds to the violet region.

More usefully, therefore, these opsins can be named S (blue), M (green) and L (red)

(Bowmaker, 1983).

The human M and L cone opsins are each 364 amino acids in length and show 96%

identity to each other. In fact they differ at only 15 residues (figure 1.9 and figure 1.6).

The degree of identity between these opsins and the S opsin is only 40%, as is the identity

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