The Regulation of Visual Transduction and Olfaction
the photoreceptors. An outward membrane current was recorded during illumination and when dim flashes of light were applied, the current fluctuated in a quantal manner. Similar quantal responses have been observed in photocurrent records from primate rod cells; examples are shown in Figure 6.1. Each unitary event is considered to be the result of an interaction between a single photon and a single pigment molecule.
Although the human eye is able to sense fluxes of just a few photons per second, it can also detect subtle intensity differences under conditions of very bright illumination. This gives it a remarkable dynamic range. Additionally, the human eye can detect transient events and recover rapidly. For example, when exposed to a train of dim flashes, we can distinguish individual
events up to a frequency of 24 Hz.The flickering image is an enduring (and endearing) feature of old movies, in which the frame rate is slower than the flicker-fusion frequency of the audience. The faster frame rate of modern cinematography (and television) is just sufficient to allow successive images to fuse and give the impression of continuous motion. The flicker-fusion frequency of other species, such as some insects, can exceed 100 Hz.
And when a Coal of Fire moved nimbly in the circumference of a Circle, makes the whole circumference appear like a Circle of Fire; is it not because the Motions excited in the bottom of the Eye by the Rays of Light are of a lasting nature, and continue till the Coal of Fire in going round returns to its former place? And considering the lastingness of the Motions excited in the bottom of the eye by Light, are they not of a vibrating nature?
Sir Isaac Newton, Opticks.
Photoreceptor mechanisms
We know more about signalling through vertebrate photoreceptors than through any of the other 7TM receptors interacting with heterotrimeric GTP-binding proteins. However, a warning is in order. It should not be imagined that what we learn here is a general reflection of G-protein- coupled receptor mechanisms. Almost everything that happens in vertebrate phototransduction is the very converse of the normal sequence of events by which receptors signal the generation of second messengers and then
their interactions with effectors. This is a world turned upside down. 11-cis-retinal is the prosthetic group in rhodopsin that absorbs visible light (see Figure 6.3). It is not only the chromophore, but also the ligand and this is already bound covalently to rhodopsin before receipt of the light signal. After photoisomerization it detaches and diffuses into a neighbouring cell. The mechanism of transduction is coupled to membrane hyperpolarization and a reduction rather than depolarization, and an elevation of the concentration
Rhodopsin, initially known as visual purple, was first isolated and described in 1878 by Willy Kühne, professor of physiology at the University of Heidelberg. He realized that its purple colour is due
to a chemical group (chromophore) distinct from those present in egg yolk and -carotene. He showed that it fades from a deep red to pink when illuminated by light of appropriate wavelengths. In addition to rhodopsin, Kühne also coined the words trypsin, enzyme, and myosin.5
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Fig 6.1 Detecting light responses in single rod cells.
(a) Responses of a single retinal rod cell to flashes of light. These evoke transient photocurrents in the membrane of a single rod cell
outer segment. The amplitude of the transient outward currents increases with intensity up to a saturating level of 34 pA (wavelength 500 nm, flash duration 11 ms, photon density 1.7–503 photons m 2, species monkey, Macaca fascicularis). (b)
Quantal responses of single retinal rod cells to dim flashes. The membrane current responses of a single rod cell to a train of dim flashes are variable in amplitude. This variability together with the presence of background noise gives the appearance of a sequence
of successes and failures. Amplitude histograms (not shown) reveal a quantal response to light. The unitary events have an average amplitude of 0.7 pA and they correspond to the detection of a single photon
(0.6 photons m 2, other conditions similar to panel a). (c) Response of a single rod cell to steady illumination. Records of photocurrents correspond to the superposition of random photon responses. The photon flux density is indicated below each trace (photonsm 2 s 1).
Adapted from Baylor et al.4
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of cytosol Ca2 . The mechanism of down-regulation and preparation of the system for a subsequent bout of illumination continues in this vein, generally the very reverse of what we have learned about hormone receptor systems. By contrast, in the invertebrate photoreceptor system we appear to be on rather more familiar ground. Here, the excitation of rhodopsin is coupled mostly through Gq to the activation of PLC and elevation of cytosol Ca2 .
Photoreceptor cells
The photoreceptor cells of the retina (illustrated in Figure 6.2) are of two types. There are the cones which collectively provide colour discrimination (photopic vision), and the rods which are responsible for sensing low levels of light (scotopic vision). The human retina contains 120 million rods and 6 million cones. It is common experience that the appearance of colour is lost when objects are viewed in dim light and this is because the image is then detected only by the rods. The mechanism of phototransduction in cones is very similar to that of rods, differing only in terms of colour specificity. There are three types of cone, each containing one of three different pigments. Each of these, like rhodopsin, consists of 11-cis-retinal embedded in an opsin molecule. The three different cone opsins tune the absorption spectrum of the attached retinal to sense either red, green, or blue light. Rods, on the other hand,
are tuned to match to the spectral distribution of dim natural light, with an optimum at 500 nm. Subsequent discussion will concentrate on rod cells.
Rod cells
Photoreceptor cells are highly differentiated epithelial cells in which the lightsensing region is segregated from the main cell body as the outer segment
Fig 6.2 Organization of mammalian retina.
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(Figure 6.3). It is a salient (if not another perverse) feature of the vertebrate visual system that the light enters these cells through the end of the cell body distal to the photosensitive outer segment. Not only this, before it enters the photoreceptors, it first traverses a network of blood vessels and then several layers of neuronal cells. The outer segments of the photoreceptors contain arrays of 1000–2000 intracellular discs, flattened vesicular structures about 16 nm thick, the membranes of which contain the photopigment, rhodopsin. Each disc is formed by the invagination of the plasma membrane to produce a structure that eventually becomes detached, so that the intradiscal space is topologically equivalent to the extracellular space. By weight, 50% of the disc membrane is protein, mostly rhodopsin. A single human rod cell contains on average 108 molecules of rhodopsin.6 This protein is organized in the same way as the conventional 7TM receptors that are found in plasma membranes. The glycosylated N-terminus projects into the intradiscal space and the C-terminal domain, containing several potential phosphorylation sites (serine/ threonine), is exposed to the cytosol.
Transducin, the G protein that transduces the light signal,7 is also very abundant, though clearly outnumbered by rhodopsin. It comprises 20% of the total (50% of the soluble) protein and unlike other G-proteins, it is
Fig 6.3 Rod photoreceptor cell and the rhodopsin molecule.
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soluble. Photoreceptor cells provide the only instance in which the number of receptors greatly exceeds the number of G protein molecules to which they couple. The need for such vast amounts of rhodopsin is probably determined by the fact that photons travel in straight lines. Unlike normal soluble ligands, they are unable to diffuse in the extracellular medium until they are captured by their favoured receptor.
The chromophore, 11-cis retinal, is coupled through its aldehyde group to the -amino group of a lysine in the centre of the G transmembrane helix, as a protonated Schiff base (Figure 6.4). Although covalent, this linkage is broken
subsequent to the photoisomerization to all-trans-retinal, which is released for reprocessing.
The extremities of the rod outer segments protrude into the layer of cells that form the pigment epithelium. This layer serves a number of purposes. Most significantly in connection with phototransduction, its cells contain the metabolic enzymes that regenerate the active 11-cis isomer from the inactive all-trans-retinal following its detachment from the visual pigment in the neighbouring rods. As the name suggests, the cells of this layer contain their own pigment, which is melanin. This absorbs light that has penetrated past
the arrays of discs in the rod cells and so prevents it from being scattered back into the photoreceptor layer. A failure to synthesize melanin (associated with the inherited conditions described as albinism) causes problems associated with bright lights and glare.
The principal enzymes of the transducing cascade are the G protein transducin (Gt) and the effector enzyme cyclic GMP phosphodiesterase. rhodopsin is an integral membrane protein, but the phosphodiesterase is soluble. Transducin is attached to the membrane but dissociates and is
Fig 6.4 Structures of retinal.
(a) 11-cis-retinal. (b) all-trans-retinal. (c) Formation of a protonated Schiff base from an aldehyde and an amine.
Beyond these difficulties, in albinism the fovea (the central region of the retina responsible for visual acuity) may also fail to develop properly, so that the eye cannot process sharp images well.
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Fig 6.5 Principal cation movements across a rod cell plasma membrane in the dark.
released into the cytosol as the rhodopsin becomes activated following illumination.8,9
The cell body contains the nucleus, mitochondria, and the protein synthetic apparatus. This is very active, enabling the photosensory cells to keep pace with the loss of the outer segment discs which have a life of just a few weeks.10 Over this period, the discs migrate progressively to the distal end of the outer segments where they are shed and phagocytosed by the pigment epithelial cells.11 The inner ends of the photosensory cells form synapses with intermediate neurons. The neurotransmitter is glutamate.
Dark current and signal amplification
In the absence of illumination, the membrane potential of the photoreceptor cells is about 30 mV.This is much less negative than the potential of excitable nerve and muscle cells and is due to a continuous photoreceptor dark current caused by an influx of Na and Ca2 through non-specific cation channels. The ions are extruded from the cells by pumps (Na , K -ATPase) and exchangers (Na /Ca2 ) which are located in the plasma membrane of the inner segment of the photoreceptor cells. Thus there is a constant flow of cations into and out of the cell (Figure 6.5).
Cyclic GMP: a second messenger in reverse
In the dark, the cation channels are held in their open configuration by the presence of cGMP. The effect of light is to initiate a series of molecular
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