Analogue pigment studies of chromophore-protein interactions in metarhodopsins

Several analogue pigments have been prepared containing retinals altered at the cyclohexyl ring or proximal to the aldehyde group in order to examine the role of the chromophore in the formation of the metarhodopsin I and II states of visual pigments. Deletion of the 13-methyl group on the isoprenoid chain did not affect metarhodopsin formation. However, analogue pigments containing chromophores with modified rings did not show the typical absorption changes associated with the metarhodopsin transitions of native or regenerated rhodopsins. In particular, 4-hydroxyretinal pigments did not show clear transitions between the metarhodopsin I and metarhodopsin II states. Pigment formed with an acyclic retinal showed no evidence by absorption spectroscopy of metarhodopsin formation. A retinal altered by substitution of a five-membered ring containing a nitroxide required a more acidic pH than the native pigment for formation of the metarhodopsin II state. ESR data suggest that the ring remains buried within the protein through the metarhodopsin II state. However, the Schiff base linkage is susceptible to hydrolysis of hydroxylamine in the metarhodopsin II state. These data indicate that (1), in the transition from rhodopsin to metarhodopsin II, major protein conformational changes are occurring near the lysine-retinal linkage whereas the ring portion of the chromophore remains deeply buried within the protein and (2) pigment absorptions characteristic of the metarhodopsin I and II states may be due to specific protein-chromophore interactions near the region of the chromophore ring.     

Cone monochromacy and visual pigment spectral tuning in wobbegong sharks

Much is known regarding the evolution of colour vision in nearly every vertebrate class, with the notable exception of the elasmobranchs. While multiple spectrally distinct cone types are found in some rays, sharks appear to possess only a single class of cone and, therefore, may be colour blind. In this study, the visual opsin genes of two wobbegong species, Orectolobus maculatus and Orectolobus ornatus, were isolated to verify the molecular basis of their monochromacy. In both species, only two opsin genes are present, RH1 (rod) and LWS (cone), which provide further evidence to support the concept that sharks possess only a single cone type. Examination of the coding sequences revealed substitutions that account for interspecific variation in the photopigment absorbance spectra, which may reflect the difference in visual ecology between these species.     

Molecular basis of spectral tuning in the newt short wavelength sensitive visual pigment

Previously we reported the sequence of the member of the short wavelength sensitive 2 (SWS2) family of vertebrate visual pigments from the retina of the Japanese common newt, Cynops pyrrhogaster[Takahashi, Y. et al. (2001) FEBS Lett. 501, 151-155]. Now we have expressed the apopigment and regenerated it with A1 retinal. Its absorption maximum, 474 nm, is greatly red shifted compared to other known SWS2 pigments (418-455 nm). To determine the amino acid residues that control its spectral tuning, we replaced the residues that were near the chromophore and which differed between the newt and the bullfrog (lambda(max) = 430 nm) wild-type SWS2 pigments: Pro91Ser, Ser94Ala, Ile122Met, Cys127Ser, Ser211Cys, Tyr261Phe, and Ala292Ser. Each of these site-directed mutants led to blue shifts of the newt pigment with five of them causing substantial shifts; their sum was about equal to the difference between the absorption maximum of the bullfrog and newt pigments, 44 nm. The 32 nm shift of the absorption maximum of the multiple seven-residue mutant to 442 nm is fairly close to that of the wild-type bullfrog pigment. Thus, the seven amino acid residues that we replaced are the major cause of the red shift of the newt SWS2 pigment's spectrum. Two of the residues, 91 and 94, have not previously been identified as wavelength regulating sites in visual pigments. One of these, 91, probably regulates color via a new mechanism: altering of a hydrogen bonding network that is connected via a water to the chromophore, in this case its counterion, Glu113.     

On the implications of bistability of visual pigment systems

The characteristics of different responses of invertebrate photoreceptors are reviewed. Invertebrate photopigment bistability has made possible the functional operational dissection of the pigment transition scheme. Outlasting the usual stimulus-coincident late receptor potential (LRP), additional antagonistic responses have been found: the prolonged depolarizing after-potential (PDA) arising from a net rhodopsin to metarhodopsin pigment shift, and a PDA-depression and an anti-PDA effect which arise from a reverse shift and cancel the PDA when induced during or closely before it. The characteristics of these aftereffects and of the LRP are reviewed, analyzed and compared. Both potentials require rhodopsin activation and they share the characteristics of a common ionic conductance-change mechanism. However, for the LRP response to weak stimuli, no antagonistic metarhodopsin-dependent effect has been found analogous to PDA-depression and the anti-PDA. However, this is just the response level where interactive effects would be weakest. For more intense stimuli, pigment-state effects on the shape of the LRP have been found, and net pigment shifts affect the strength of a facilitatory effect.Based on material presented at the European Neurosciences Meeting, Florence, September 1978     

On the disulphide bonds of rhodopsins.

Carboxymethylation using 14C- or 3H-labelled iodoacetic acid has been used to identify the cysteine residues in bovine rhodopsin involved in the formation of the two intramolecular disulphide bridges. Iodo[2-14C]acetic acid was used to modify 5.8-5.9 residues of cysteine under non-reducing conditions. After dialysis and reduction of disulphide bridges by 2-mercaptoethanol, iodo[2-3H]acetic acid was employed to covalently modify 3.3-3.6 residues of cysteine. Peptide purification and sequencing has unambiguously shown that cysteine residues 322 and 323 are only carboxymethylated after reduction of disulphide bridges. Indirect evidence presented, now coupled with the earlier finding [Findlay & Pappin (1986) Biochem. J. 238, 625-642] suggests that the other disulphide bridge is formed between cysteine residues 110 and 187. A comparison is made of all the sequences of mammalian rhodopsins and colour pigments and attention is drawn to the fact that whereas Cys-322 and Cys-323 are conserved only in three rhodopsins (bovine, ovine and human), the residues corresponding to Cys-110 and Cys-187 are found in all the visual proteins (from rods as well as human cones).     

Metarhodopsin control by arrestin,light-filtering screening pigments,and visual pigment turnover in invertebrate microvillar photoreceptors

The visual pigments of most invertebrate photoreceptors have two thermostable photo-interconvertible states, the ground state rhodopsin and photo-activated metarhodopsin, which triggers the phototransduction cascade until it binds arrestin. The ratio of the two states in photoequilibrium is determined by their absorbance spectra and the effective spectral distribution of illumination. Calculations indicate that metarhodopsin levels in fly photoreceptors are maintained below ~35% in normal diurnal environments, due to the combination of a blue-green rhodopsin, an orange-absorbing metarhodopsin and red transparent screening pigments. Slow metarhodopsin degradation and rhodopsin regeneration processes further subserve visual pigment maintenance. In most insect eyes, where the majority of photoreceptors have green-absorbing rhodopsins and blue-absorbing metarhodopsins, natural illuminants are predicted to create metarhodopsin levels greater than 60% at high intensities. However, fast metarhodopsin decay and rhodopsin regeneration also play an important role in controlling metarhodopsin in green receptors, resulting in a high rhodopsin content at low light intensities and a reduced overall visual pigment content in bright light. A simple model for the visual pigment–arrestin cycle is used to illustrate the dependence of the visual pigment population states on light intensity, arrestin levels and pigment turnover.     

Genetic basis of spectral tuning in the violet-sensitive visual pigment of African clawed frog, Xenopus laevis

Ultraviolet (UV) and violet vision in vertebrates is mediated by UV and violet visual pigments that absorb light maximally (lambdamax) at approximately 360 and 390-440 nm, respectively. So far, a total of 11 amino acid sites only in transmembrane (TM) helices I-III are known to be involved in the functional differentiation of these short wavelength-sensitive type 1 (SWS1) pigments. Here, we have constructed chimeric pigments between the violet pigment of African clawed frog (Xenopus laevis) and its ancestral UV pigment. The results show that not only are the absorption spectra of these pigments modulated strongly by amino acids in TM I-VII, but also, for unknown reasons, the overall effect of amino acid changes in TM IV-VII on the lambdamax-shift is abolished. The spectral tuning of the contemporary frog pigment is explained by amino acid replacements F86M, V91I, T93P, V109A, E113D, L116V, and S118T, in which V91I and V109A are previously unknown, increasing the total number of critical amino acid sites that are involved in the spectral tuning of SWS1 pigments in vertebrates to 13.     

The visual pigment and visual cycle of the lobster,Homarus

Summary The visual pigment of the American lobster,Homarus americanus, has been studied in individual isolated rhabdoms by microspectrophotometry. Lobster rhodopsin has _max at 515 nm and is converted by light to a stable metarhodopsin with _max at 490 nm. These figures are in good agreement with corresponding values obtained by Wald and Hubbard (1957) in digitonin extracts. Photoregeneration of rhodopsin to metarhodopsin is also observed. The absorbance spectrum of lobster metarhodopsin is invariant with pH in the range 5.4–9, indicating that even after isomerization of the chromophore fromcis totrans, the binding site of the chromophore remains sequestered from the solvent environment. Total axial density of the lobster rhabdom to unpolarized light is about 0.7.As described for several other Crustacea, aldehyde fixation renders the metarhodopsin susceptible to photobleaching, a process that is faster at alkaline than at neutral or acid pH. Small amounts of a photoproduct with _max at 370 nm are occasionally seen. A slower dark bleaching of lobster rhabdoms (_1/2–2 h) also occurs, frequently through intermediates with absorption similar to metarhodopsin.The molar extinction coefficient of metarhodopsin is about 1.2 times greater than that of rhodopsin, each measured at their respective _max. Isomerization of the chromophore fromcis totrans is accompanied by a change in the orientation of the absorption vector of about 3°. The absorption vector of metarhodopsin is either tilted more steeply into the membrane or is less tightly oriented with respect to the microvillar axes.When living lobsters are kept at room temperature, light adaptation does not result in an accumulation of metarhodopsin. At 4 °C, however, the same adapting lights cause a reduction of rhodopsin and an increase in metarhodopsin. There is thus a temperature-sensitive regeneration mechanism that supplements photoregeneration. Following 1 ms, 0.1 joule xenon flashes that convert about 70% of the rhodopsin to metarhodopsin in vivo, dark regeneration occurs in the living eye with half-times of about 25 and 55 min at 22 °C and 15 °C respectively.This work was supported by USPHS research grant EY 00222 to Yale University. S.N.B. was aided by NIH Postdoctoral Fellowship EY 52378.     

The nature of the gecko visual pigment

Retinal extracts of the Australian gecko, Phyllurus milii (White), have revealed the presence of a photosensitive pigment, unusual for terrestrial animals, because of its absorption maximum at 524 mµ. This pigment has an absorption spectrum which is identical in form with that of other visual chromoproteins. It is not a porphyropsin, for bleaching revealed the presence, not of retinene₂, but of retinene₁ as a chromophore. Photolabile pigments with characteristics similar to those of the Phyllurus visual pigment were also detected in retinal extracts of six other species of nocturnal geckos. The presence of this retinal chromoprotein adequately accounts for the unusual visual sensitivity curve described by Denton for the nocturnal gecko. This pigment may have special biological significance in terms of the unique phylogenetic position of geckos as living representatives of nocturnal animals which retain some of the characteristics of their diurnal ancestors. The occurrence of this retinene₁ pigment, intermediate in spectral position between rhodopsin and iodopsin, is interpreted in support of the transmutation theory of Walls. The results and interpretation of this investigation point up the fact that, from a phylogenetic point of view, too great an emphasis on the duplicity theory may serve to detract attention from the evolutionary history of the retina and the essential unitarianism of the visual cells.     

The nature of the lamprey visual pigment

From the retina of the land-locked population of the sea lamprey, Petromyzon marinus, a photolabile pigment was extracted which was identified spectrophotometrically as a member of the rhodopsin group of pigments. Using the absorption spectrum of a relatively pure solution and analysis by means of difference spectra, the peak of this pigment was placed at about 497 mµ. The method of selective bleaching by light of different wave lengths revealed no significant amounts of any other pigment in the extracts. A similar pigment was also detected in retinal extracts of the Pacific Coast lamprey, Entospenus tridentatus. These results are significant for two reasons: (a) the lamprey is shown to be an example of an animal which spawns in fresh water but which is characterized by the presence of rhodopsin, rather than porphyropsin, in the retina; (b) the primitive phylogenetic position of the lamprey suggests that rhodopsin was the visual pigment of the original vertebrates.     

On some mathematical techniques for the analysis of visual spectral sensitivities.

Starting from known spectral properties of visual photopigments and photoreceptors, a mathematical construction of neural response functions to visual stimuli is obtained. Included in this is a somewhat general derivation of the univariance principle. Temporal dependence of response on stimulus is included in the formulation. Special attention is given to the case of flash stimuli and their resulting spectral sensitivities. This formulation is applied to certain physiological spectral sensitivity measurements that are at variance with known spectrophometric results. An analysis of the data in these cases suggests that they correspond to "pseudo-pigments" arising from the neural interaction of several photopigments. The method of analysis is constructive and identifies the gamma-max's of the interacting photopigments. These are found to be in good agreement with existing spectrophotometric measurements.     

A novel amino acid substitution is responsible for spectral tuning in a rodent violet-sensitive visual pigment

Cone short-wave (SWS1) visual pigments can be divided into two categories that correlate with spectral sensitivity, violet sensitive above 390 nm and ultraviolet sensitive below that wavelength. The evolution and mechanism of spectral tuning of SWS1 opsins are proving more complex than those of other opsin classes. Violet-sensitive pigments probably evolved from an ancestral ultraviolet-sensitive opsin, although in birds ultraviolet sensitivity has re-evolved from violet-sensitive pigments. In certain mammals, a single substitution involving the gain of a polar residue can switch sensitivity from ultraviolet to violet sensitivity, but where such a change is not involved, several substitutions may be required to effect the switch. The guinea pig, Cavia porcellus, is a hystricognathous rodent, a distinct suborder from the Sciurognathi, such as rats and mice. It has been shown by microspectrophotometry to have two cone visual pigments at 530 and 400 nm. We have ascertained the sequence of the short-wave pigment and confirmed its violet sensitivity by expression and reconstitution of the pigment in vitro. Moreover, we have shown by site-directed mutagenesis that a single residue is responsible for wavelength tuning of spectral sensitivity, a Val86Phe causing a 60 nm short-wave shift into the ultraviolet and a Val86Tyr substitution shifting the pigment 8 nm long wave. The convergent evolution of this mammalian VS pigment provides insight into the mechanism of tuning between the violet and UV.     

Magnetic anisotropy of the visual pigment rhodopsin.

A new estimate of diamagnetic anisotropy of the frog rhodopsin is reported. The estimate is obtained by combining the data of magnetic field induced orientation of isolated frog rod outer segments as measured by Chagneux and Chalazonitis (1972) and the data of diamagnetic anisotropy of lecithin membranes as recently reported by Boroske and Helfrich (1978). The anisotropy of the volume susceptibilities of frog rhodopsin is calculated to be 4.4 X 10(-8) cgs unit/cm3, which corresponds to 1.5 X 10(-27) cgs unit/molecule, or 9.0 X 10(-4) cgs unit/mol.     

Molecular physiology of visual pigment rhodopsin

The key physiological functions of the rhodopsin molecule are reviewed. Molecular mechanisms of visual pigments spectral tuning, photoisomerization of the 11-cis-retinal chromophore that triggers the phototransduction process, formation of physiologically active state of rhodopsin as a G-protein-coupled receptor, rhodopsin visual cycle, and consequences of its impairment are evaluated. Visual pigment rhodopsin performs several functions, providing spectral sensitivity of photoreceptor cells, phototransduction processes and light and dark adaptation. Genetically determined defects of visual pigment molecule and proteins involved into mechanisms of phototransduction and adaptation or into mechanism of visual cycle are directly linked to pathogenesis of different forms of degenerative retina diseases. Understanding the molecular mechanisms of these physiological processes uncovers the way to direct investigation of pathogenesis of these severe eye diseases.     

The kinetics of visual pigment systems

Using early receptor potential (ERP) measurements, we show that the bistable pigment in the barnacle photoreceptor behaves according to the conclusions of the preceding article (Hochstein et al., 1978): (1) The populations of both stable states approach their steady-state or saturation values under steady illumination exponentially with the same rate constant; the wavelength dependence of this rate constant is called the relaxation spectrum. — (2) The saturation values are independent of initial population and of light intensity; the wavelength dependence of the saturation population is called the saturation spectrum. — (3) The measured relaxation and saturation spectra agree with those calculated, by the theory of the preceding article, from the experimentally determined transition parameters of the pigment system. — We then demonstrate the applicability of relaxation and saturation measurements to the question of whether a single bi-stable pigment system serves, or two or more separate systems serve, as the origins(s) of the ERP and of other phenomena observed in the barnacle photoreceptor: The prolonged depolarizing afterpotential (PDA) and its depression and prevention (anti-PDA). By showing that the relaxation spectra for these phenomena match one another and that of the ERP, and that the same is true for the saturation spectra, we demonstrate that these phenomena originate from the same single bi-stable pigment system as the ERP.     

Receptor-coupling properties of the invertebrate visual guanine nucleotide binding protein iGqalpha

Invertebrate visual iG(q)alpha is homologous to mammalian mG(q)alpha in two of the three domains important for G protein interaction with receptors; the C-terminus and the linker regions that connect the helical and ras-like domains of the alpha subunit. The third receptor-interacting domain, the N-terminus, contains a six amino acid extension MTLESI in mG(q)alpha that is not present in iG(q)alpha. In co-expression studies we assessed the promiscuity and efficacy of receptor coupling to phospholipase C (PLC) by iG(q)alpha, a non-palmitoylated mutant iG(q)alpha(C3,4A), mG(q)alpha and G(15)alpha. The invertebrate G proteins and mG(q)alpha only coupled to G(q)-coupled receptors (m1 muscarinic acetylcholine receptor (mChR1), alpha(1A)-adrenergic receptor (alpha1-AR)) and not to the G(i/s)-coupled receptors (CCR1 receptor, beta2-adrenergic receptor or dopamine D1 receptor) while G(15)alpha coupled to all receptors. iG(q)alpha and iG(q)alpha(C3,4A) both had double the efficacy for PLC activation compared to the mammalian G proteins when co-expressed with mChR1 and alpha1-AR. The increased efficacy of iG(q)alpha compared to mG(q)alpha was also seen downstream of PLC with carbachol stimulation of the mitogen-activated protein kinase, ERK1/2. Addition of the MTLESI extension onto the N-terminus of iG(q)alpha decreased its efficacy by 35% whereas deletion of this sequence from mG(q)alpha increased its efficacy by 60% in the PLC and ERK1/2 assays. iG(q)alpha, iG(q)alpha(C3,4A) and mG(q)alpha all displayed similar receptor-independent AlF(4)(-)activation of PLC and guanosine triphosphate hydrolysis (GTPase) activity. iG(q)alpha, and iG(q)alpha(C3,4A) both had increased receptor-activated guanosine 5'-[gamma-[(35)S]thio]triphosphate ([(35)S]GTPgammaS) binding when compared to mG(q)alpha when co-expressed with the mChR1. These results demonstrate that G(q) protein efficacy is at least partially determined by the presence of the amino-terminal MTLESI extension. Comparison of [(35)S]GTPgammaS binding rates helps explain the increased efficacy of the invertebrate G proteins.