Binding of retinaldehyde to the protein opsin in the rods, and related proteins in the cones, of the retina gives a highly sensitive signal transduction and amplification system, such that a single photon results in a measurable change in the current across the outer section membrane, and hence the propagation of a nerve impulse. In the outer segment of rod cells, opsin may constitute more than 90% of the total protein; it is present at a concentration of approximately 3 mmol per L.
Photoexcited rhodopsin activates transducin, a G-protein, which in turn stimulates cyclic GMP phosphodiesterase; this leads to closing of an ion channel, hyperpolarization of the membrane, and a decreased rate of neurotransmitter release (Wald, 1968; Stryer, 1986; Chabré and Deterre, 1989).
The pigment epithelium of the retina receives all- trans-retinol fromplasma RBP. It is then isomerized to 11-ds-retinol, which may either be stored as 11-ds-retinyl esters or oxidized to 11-ci's-retinaldehyde, which is transported to the photoreceptor cells bound to an interphotoreceptor retinoid binding protein.
As shown in Figure 2.5, within the rods and cones of the retina, 11-cis-retinaldehyde forms a protonated Schiff base to the e-amino group of lysine296 in opsin, forming the holo-protein rhodopsin. Lysine296 is within the membrane, in one of the transmembrane helical regions of the protein. Opsins are cell type-specific. They serve to shift the absorption of 11-cis-retinaldehyde from ultraviolet (UV) light into what we call, in consequence, the visible range -either a relatively broad spectrum of sensitivity for vision in dim light (in the rods, with an absorbance peak at 500 nm) or more defined spectral peaks for differentiation of colors in stronger light (in the cones). The absorption maxima are at 425 (blue), 530 (green), or 560 nm (red), depending on the cell type.
Any one cone cell contains only one type of opsin and is sensitive to only one color of light. Color blindness results from loss or mutation of one or the other of the cone opsins. The combination of 11-cis-retinaldehyde with cone opsin is sometimes called iodopsin, with rhodopsin meaning more specifically the holo-protein of rod opsin. Most studies of the mechanisms of vision shown in Figure 2.5 have been performed using rods; by extrapolation, it is assumed that the same mechanisms are involved in cone vision.
Opsin can be considered to be a retinaldehyde receptor protein, functioning in the same way as cell surface receptor G-proteins (Sakmar, 1998). Like receptor proteins, opsin is a transmembrane protein with seven a -helical regions in the transmembrane domain; the difference is that opsin spans the intracellular disk membrane of the rod or cone cell, whereas hormone and neurotransmit-ter receptors span the plasma membrane of the cell. The response time of rhodopsin is considerably faster than that of cell surface receptor proteins.
The absorption of light by rhodopsin results in a change in the configuration of the retinaldehyde from the 11-cis to the all- trans isomer, together with a conformational change in opsin. This results in both the release of retinaldehyde from the Schiff base and the initiation of a nerve impulse. The overall process is known as bleaching, because it results in the loss of the color of rhodopsin.
The formation of the initial excited form of rhodopsin - bathorhodopsin -depends on the isomerization of 11-cis-retinaldehyde to a strained form of all- trans-retinaldehyde. This occurs within picoseconds of illumination and is the only light-dependent step in the visual cycle. Thereafter, there is a series of conformational changes leading to the formation of metarhodopsin II. In metarhodopsin II, the Schiff base is unprotonated, and the retinaldehyde is in the unstrained all- trans configuration.
The conversion of metarhodopsin II to metarhodopsin III is relatively slow, with a time course of minutes. It is the result of phosphorylation of serine residues in the protein catalyzed by rhodopsin kinase. The final step is hydrolysis to release all-frans-retinaldehyde and opsin.
Under conditions of low light intensity, the all-frans-retinaldehyde released from rhodopsin is reduced to all-frans-retinol, which is then transported to the retinal pigment epithelium bound to the interphotoreceptor RBP. This protein also binds fatty acids, including palmitate and docosa-hexaenoic acid (C22:6 «3), which is known to be essential for vision and which comprises some 50% of the phospholipid of photoreceptor cells.
In the retinal pigment epithelium, palmitate is bound to the fatty acid binding site of the interphotoreceptor RBP, and the retinoid binding site has a high affinity for ll-ds-retinaldehyde, which is to be transported to the photoreceptor cells. In the photoreceptor cells, the palmitate is displaced by docosahex-aenoic acid, which causes a conformational change in the protein, so that it no longer binds ll-ds-retinaldehyde, which is delivered to the photoreceptor cells and binds all-frans-retinol for transport back to the pigment epithelium. Here, the docosahexaenoic acid is displaced by palmitate, and the affinity of the protein for ll-ds-retinaldehyde is restored (Palczewski and Saari, 1997; Tschanz and Noy, 1997).
Under conditions of high light intensity, all-frans-retinaldehyde binds to the retinal G-protein-coupled receptor (RGR), which catalyzes photoisomer-ization to ll-ds-retinaldehyde - the reverse of the photoisomerization catalyzed by rhodopsin. Retinaldehyde dehydrogenase binds to the RGR and reduces the ll-ds-retinaldehyde to ll-ds-retinol, which then enters the pool available to undergo oxidation to the aldehyde and reform rhodopsin. Knockout mice lacking RGR have impaired responses to light under conditions of continuous intense illumination, but normal responses under conditions of low-light intensity when all-frans-retinaldehyde is reduced to all-frans-retinol and transported by the interphotoreceptor retinoid binding protein (Hao and Fong, l999; Chen et al., 200l).
Metarhodopsin II is the excited form of rhodopsin that initiates the gua-nine nucleotide amplification cascade that causes nerve stimulation. The final event is a hyperpolarization of the outer section membrane of the rod or cone caused by the closure of sodium and calcium channels through the membrane - excitation of a single molecule of rhodopsin, the action of a single photon, causes a drop of l pA in the normal dark current across this membrane in rods. In cones, the response to a single photon is only 1% to 10% of that in the rods.
In the dark, the sodium channels are kept open, and there is a dark current because they bind GMP. They are closed by the loss of this bound GMP as it is hydrolyzed to 5 -GMP by phosphodiesterase. The phosphodiesterase is activated by the guanine nucleotide binding protein (G-protein) transducin.
Transducin, in its inactive form in the dark, has bound GDP; interaction with metarhodopsin II causes it to release this GDP and bind GTP. Transducin-GTP has a low affinity for metarhodopsin II, which is therefore free to interact with another molecule of transducin-GDP. Thus, for as long as metarhodopsin II remains in its active state (i.e., until it has been fully phosphorylated and converted to metarhodopsin III, which does not interact with transducin-GDP), it will continue to activate transducin molecules.
Metarhodopsin II activates transducin, leading to an exchange of bound GDP for GTP; several hundred molecules of transducin are activated by a single molecule of metarhodopsin II within a fraction of a second. Transducin-GTP binds to, and activates, GMP phosphodiesterase, lowering the intracellular concentration of cGMP. As cGMP falls, a cation channel in the membrane closes, thus interrupting the steady inward current of sodium and calcium ions. This leads to hyperpolarization of the membrane and reduced secretion of neurotransmitter (Baylor, 1996).
Like other G-proteins, transducin has innate GTPase activity, and over a time course of seconds or less is autocatalytically converted to transducin-GDP, which does not interact with phosphodiesterase. This restores the normal inhibition of phosphodiesterase, permitting cGMP concentrations to rise again, reopening the sodium channels and restoring the dark current.
Metarhodopsin II is inactivated by phosphorylation of three serine residues at the carboxyl terminal of the protein, catalyzed by rhodopsin kinase. In transgenic mice with carboxyl terminal-truncated rhodopsin, lacking the phosphorylation sites, there is a prolonged response to a single photon. Rhodopsin kinase is activated by its substrate, metarhodopsin II, and is inhibited by calcium bound to the protein recoverin, which thus prolongs the photoresponse.
Phosphorylation is a necessary, but not sufficient, condition for quenching metarhodopsin II; it also has to bind the protein arrestin before it loses the bound retinaldehyde and is converted to metarhodopsin III. Then, it is de-phosphorylated by protein phosphatase 2A and a calcium-activated protein phosphatase. It is this dephosphorylation of metarhodopsin III that is correlated with dark adaptation and regeneration of active rhodopsin by binding to
11-ds-retinaldehyde (Palczewski and Saari, 1997; Hurley etal., 1998; Kennedy etal., 2001).
The rate-limiting step in initiation of the visual cycle is the regeneration of 11-ds-retinaldehyde. In vitamin A deficiency, when there is little 11-ds-retinyl ester in the pigment epithelium, both the time taken to adapt to darkness and the ability to see in poor light will be impaired.
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