Cyclic nucleotide-gated ion channels in rod photoreceptors are protected from retinoid inhibition

In vertebrate rods, photoisomerization of the 11-cis retinal chromophore of rhodopsin to the all-trans conformation initiates a biochemical cascade that closes cGMP-gated channels and hyperpolarizes the cell. All-trans retinal is reduced to retinol and then removed to the pigment epithelium. The pigment epithelium supplies fresh 11-cis retinal to regenerate rhodopsin. The recent discovery that tens of nanomolar retinal inhibits cloned cGMP-gated channels at low [cGMP] raised the question of whether retinoid traffic across the plasma membrane of the rod might participate in the signaling of light. Native channels in excised patches from rods were very sensitive to retinoid inhibition. Perfusion of intact rods with exogenous 9- or 11-cis retinal closed cGMP-gated channels but required higher than expected concentrations. Channels reopened after perfusing the rod with cellular retinoid binding protein II. PDE activity, flash response kinetics, and relative sensitivity were unchanged, ruling out pharmacological activation of the phototransduction cascade. Bleaching of rhodopsin to create all-trans retinal and retinol inside the rod did not produce any measurable channel inhibition. Exposure of a bleached rod to 9- or 11-cis retinal did not elicit channel inhibition during the period of rhodopsin regeneration. Microspectrophotometric measurements showed that exogenous 9- or 11-cis retinal rapidly cross the plasma membrane of bleached rods and regenerate their rhodopsin. Although dark-adapted rods could also take up large quantities of 9-cis retinal, which they converted to retinol, the time course was slow. Apparently cGMP-gated channels in intact rods are protected from the inhibitory effects of retinoids that cross the plasma membrane by a large-capacity buffer. Opsin, with its chromophore binding pocket occupied (rhodopsin) or vacant, may be an important component. Exceptionally high retinoid levels, e.g., associated with some retinal degenerations, could overcome the buffer, however, and impair sensitivity or delay the recovery after exposure to bright light.     

Utilization of retinoids in the bullfrog retina

The capacity to generate 11-cis retinal from retinoids arising naturally in the eye was examined in the retina of the bullfrog, Rana catesbeiana. Retinoids, co-suspended with phosphatidylcholine, were applied topically to the photoreceptor surface of the isolated retina after substantial bleaching of the native visual pigment. The increase in photoreceptor sensitivity associated with the formation of rhodopsin, used as an assay for the appearance of 11-cis retinal in the receptors, was analyzed by extracellular measurement of the photoreceptor potential; in separate experiments using the isolated retina or receptor outer segment preparations, the formation of rhodopsin was measured spectrophotometrically. Treatments with the 11- cis isomers of retinal and retinol induced significant increases in both the rhodopsin content and photic sensitivity of previously bleached receptors. The all-trans isomers of retinyl palmitate, retinol, and retinal, as well as the 11-cis isomer of retinyl palmitate, were inactive by both the electrophysiological and spectrophotometric criteria for the generation of rhodopsin. Treatment with any one of the "inactive" retinoids did not abolish the capacity of subsequently applied 11-cis retinal or 11-cis retinol to promote the formation of rhodopsin. The data are discussed in relation to the interconversions of retinoids ("visual cycle of vitamin A") thought to mediate the regeneration of rhodopsin in vivo after extensive bleaching.     

Isomeric composition of retinal chromophore in dark-adapted bacteriorhodopsin

1. Retinal isomers extracted from the acid-hydrolysate of cetyltrimethylammonium bromide-treated dark-adapted bacteriorhodopsin (bRD) were analyzed in a high performance liquid chromatograph (HPLC) system. The extract from bRD contains almost equal molar amounts of both 13-cis retinal and all-trans retinal isomers. The extent of isomerization and the yield of both isomers during the isolation process were investigated by the application of the same extraction procedure to artificial bacteriorhodopsin reconstituted with 13-cis retinal isomer (13-cis bacteriorhodopsin) and also to light-adapted bacteriorhodopsin (bRL) which has been shown to contain only the all-trans isomer (all-trans bacteriorhodopsin). 2. A reconstituted bacteriorhodopsin, which had been prepared from apo-bacteriorhodopsin and an equimolar mixture of both 13-cis retinal and all-trans retinal isomers, showed an absorption spectrum having the same maximum wavelength as that of bRD even at the beginning of the reconstitution process. 3. Analysis of the photosteady states of bRD at -190 degrees C revealed that it was composed of two different species, one having 13-cis retinal and the other having all-trans retinal isomers in approximately equal molar amounts. These two also gave their respective photoproducts. 4. From these results it can be concluded that bRD contains both 13-cis retinal and all-trans retinal isomers in nearly equal molar amounts as its chromophore.     

Substitution of Pro206 and Ser86 residues in the retinal binding pocket of Anabaena sensory rhodopsin is not sufficient for proton pumping function

Anabaena sensory rhodopsin is a seven transmembrane protein that uses all-trans/13-cis retinal as a chromophore. About 22 residues in the retinal-binding pocket of microbial rhodopsins are conserved and important to control the quality of absorbing light and the function of ion transport or sensory transduction. The absorption maximum is 550 nm in the presence of all-trans retinal at dark. Here, we mutated Pro206 to Glu or Asp, of which the residue is conserved as Asp among all other microbial rhodopsins, and the absorption maximum and pKa of the proton acceptor group were measured by absorption spectroscopy at various pHs. Anabaena rhodopsin was expressed best in Escherichia coli in the absence of extra leader sequence when exogenous all-trans retinal was added. The wild-type Anabaena rhodopsin showed small absorption maximum changes between pH 4 and 11. In addition, Pro206Asp showed 46 nm blue-shift at pH 7.0. Pro206Glu or Asp may change the contribution to the electron distribution of the retinal that is involved in the major role of color tuning for this pigment. The critical residue Ser86 (Asp 96 position in bacteriorhodopsin: proton donor) for the pumping activity was replaced with Asp, but it did not change the proton pumping activity of Anabaena rhodopsin.     

Changes in the protonation state of bacterio-opsin during reconstitution of bacteriorhodopsin

Protonation changes of the protein occur during the reconstitution of bacteriorhodopsin from bacterio-opsin and all-trans retinal in the purple membrane of Halobacterium halobium. The protonation changes are conveniently determined from measures of the pH changes after photoisomerisation of 9-cis retinal in apomembrane preparations, which induces the reconstitution. In addition, to the omega-amino group of the lysine which is involved in the condensation of retinal and bacterio-opsin, the dissociation equilibria of at least two other amino acid residues are changed during the reconstitution. The results are consistent with a proposed model of chromophore structure in which an interaction of the Schiff's base occurs with two protonable amino acid residues.     

Heterogeneity in regeneration of bacteriorhodopsin from bacterio-opsin and all-trans retinal at high temperatures: implications for dynamic structural fluctuations

Measurements of regeneration kinetics were performed in order to investigate the regeneration mechanisms of bacteriorhodopsin (bR) from thermally unfolded bacterio-opsin (bO) and all-trans retinal. Regeneration kinetics data were successfully fitted to a single exponential function when regeneration was performed at 25 degrees C after incubation at high temperatures. Conversely, the process of regeneration after the addition of retinal to bO at high temperatures occurred at two different rate constants. These findings strongly suggest that the slower regeneration of bR at high temperatures occurs as a result of dynamic structural fluctuation of bO, whereas the faster process corresponds to regeneration from bO, which retains a native structure capable of retinal binding.     

The photoconversion of lumirhodopsin at 77 degrees K. Estimation of the quantum efficiency.

Evidence is presented that lumirhodopsin (containing all-trans retinal) is not directly photoconverted to bathorhodopsin (all-trans) at 77 degrees K as previously suggested (Yoshizawa and Wald. 1963. Nature (Lond.) 197:1279-1286). Rather, lumirhodopsin is converted to a new species, L' (11-cis and/or 9-cis retinal) which, on warming to room temperature, is indistinguishable from rhodopsin or isorhodopsin. The quantum efficiency for the conversion of lumirhodopsin to L' is estimated to be 0.5 +/- 0.1. This value is significantly higher than that of other all-trans to cis conversions for bovine rhodopsin intermediates, indicating that the opsin conformation has a significant effect on a pigment's quantum efficiency.     

Conformational change in bacterio-opsin on binding to retinal.

The detailed mechanism of retinal binding to bacterio-opsin is important to understanding retinal pigment formation as well as to the process of membrane protein folding. We have measured the temperature dependence of bacteriorhodopsin formation from bacterio-opsin and all-trans retinal. An Arrhenius plot of the apparent second-order rate constants gives an activation energy of 11.6 +/- 0.7 kcal/mol and an activation entropy of -4 +/- 2 cal/mol deg. Comparison of the activation entropy to model compound reactions suggests that chromophore formation in bacteriorhodopsin involves a substantial protein conformational change. Cleavage of the polypeptide chain between residues 71 and 72 has little effect on the activation energy or entropy, indicating that the connecting loop between helices B and C is not involved in this conformational change.     

Regeneration of bacteriorhodopsin from thermally unfolded bacterio-opsin and all-trans retinal at high temperatures

The temperature dependence of regeneration of bacteriorhodopsin (bR) from its apoprotein, bacterio-opsin (bO), and all-trans retinal was investigated using two different procedures to probe the structural properties of bO at high temperatures. Regeneration experiments performed at 25 degrees C after incubation of bO within the temperature range of 35-75 degrees C indicate that irreversible thermal unfolding begins at 50 degrees C. When bO is incubated for one hour and mixed with retinal at the same elevated temperatures, however, a greater extent of regeneration to bR occurs, even at temperatures ranging from 50 to 65 degrees C. These experimental results indicate that regeneration of bR occurs from thermally unfolded bO and suggest dynamic structural fluctuation of bO in the unfolded state.     

The role of retinal photoisomerase in the visual cycle of the honeybee

The compound eye of the honeybee has previously been shown to contain a soluble retinal photoisomerase which, in vitro, is able to catalyze stereospecifically the photoconversion of all-trans retinal to 11-cis retinal. In this study we combine in vivo and in vitro techniques to demonstrate how the retinal photoisomerase is involved in the visual cycle, creating 11-cis retinal for the generation of visual pigment. Honeybees have approximately 2.5 pmol/eye of retinal associated with visual pigments, but larger amounts (4-12 pmol/eye) of both retinal and retinol bound to soluble proteins. When bees are dark adapted for 24 h or longer, greater than 80% of the endogenous retinal, mostly in the all-trans configuration, is associated with the retinal photoisomerase. On exposure to blue light the retinal is isomerized to 11-cis, which makes it available to an alcohol dehydrogenase. Most of it is then reduced to 11-cis retinol. The retinol is not esterified and remains associated with a soluble protein, serving as a reservoir of 11-cis retinoid available for renewal of visual pigment. Alternatively, 11-cis retinal can be transferred directly to opsin to regenerate rhodopsin, as shown by synthesis of rhodopsin in bleached frog rod outer segments. This retinaldehyde cycle from the honeybee is the third to be described. It appears very similar to the system in another group of arthropods, flies, and differs from the isomerization processes in vertebrates and cephalopod mollusks.     

Retinyl esters are the substrate for isomerohydrolase

Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.     

Characterization of the chromophore of the third rhodopsin-like pigment of Halobacterium halobium and its photoproduct.

Halobacterium halobium contains at least three retinal-containing pigments: bacteriorhodopsin, halorhodopsin, and a third rhodopsin-like pigment (tR) absorbing at approximately 590 nm, tR590. Illumination of tR590 gives rise to a very long-lived blue absorbing photoproduct, tR370. Using high-performance liquid chromatography we show that the chromophore of tR590 is primarily all-trans retinal and its conversion by light to tR370 causes the chromophore to isomerize primarily to the 13-cis conformation. Irradiation of the tR370 gives rise to a transient photoproduct absorbing at approximately 520 nm that decays back to the initial pigment tR590. In addition to all-trans retinal, the apomembrane of tR can also combine with 13-cis retinal but not with the 9- or 11-cis isomers.     

Vibrational analysis of the all-trans retinal protonated Schiff base.

We have obtained Raman spectra of a series of all-trans retinal protonated Schiff-base isotopic derivatives. 13C-substitutions were made at the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 positions while deuteration was performed at position 15. Based on the isotopic shifts, the observed C--C stretching vibrations in the 1,100-1,400 cm-1 fingerprint region are assigned. Normal mode calculations using a modified Urey-Bradley force field have been refined to reproduce the observed frequencies and isotopic shifts. Comparison with fingerprint assignments of all-trans retinal and its unprotonated Schiff base shows that the major effect of Schiff-base formation is a shift of the C14--C15 stretch from 1,111 cm-1 in the aldehyde to approximately 1,163 cm-1 in the Shiff base. This shift is attributed to the increased C14--C15 bond order that results from the reduced electronegativity of the Schiff-base nitrogen compared with the aldehyde oxygen. Protonation of the Schiff base increases pi-electron delocalization, causing a 6 to 16 cm-1 frequency increase of the normal modes involving the C8--C9, C10--C11, C12--C13, and C14--C15 stretches. Comparison of the protonated Schiff base Raman spectrum with that of light-adapted bacteriorhodopsin (BR568) shows that incorporation of the all-trans protonated Schiff base into bacterio-opsin produces an additional approximately 10 cm-1 increase of each C--C stretching frequency as a result of protein-induced pi-electron delocalization. Importantly, the frequency ordering and spacing of the C--C stretches in BR568 is the same as that found in the protonated Schiff base.     

Photoreactions of metarhodopsin III

Meta III is an inactive intermediate thermally formed following light activation of the visual pigment rhodopsin. It is produced from the Meta I/Meta II photoproduct equilibrium of rhodopsin by a thermal isomerization of the protonated Schiff base C=N bond of Meta I, and its chromophore configuration is therefore all-trans 15-syn. In contrast to the dark state of rhodopsin, which catalyzes exclusively the cis to trans isomerization of the C11=C12 bond of its 11-cis 15-anti chromophore, Meta III does not acquire this photoreaction specificity. Instead, it allows for light-dependent syn to anti isomerization of the C15=N bond of the protonated Schiff base, yielding Meta II, and for trans to cis isomerizations of C11=C12 and C9=C10 of the retinal polyene, as shown by FTIR spectroscopy. The 11-cis and 9-cis 15-syn isomers produced by the latter two reactions are not stable, decaying on the time scale of few seconds to dark state rhodopsin and isorhodopsin by thermal C15=N isomerization, as indicated by time-resolved FTIR methods. Flash photolysis of Meta III produces therefore Meta II, dark state rhodopsin, and isorhodopsin. Under continuous illumination, the latter two (or its unstable precursors) are converted as well to Meta II by presumably two different mechanisms.     

Interphotoreceptor retinoid-binding protein is the physiologically relevant carrier that removes retinol from rod photoreceptor outer segments

Light detection by vertebrate rod photoreceptor outer segments results in the destruction of the visual pigment, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. The regeneration of rhodopsin is necessary for vision and begins with the release of the all-trans retinal and its reduction to all-trans retinol. Retinol is then transported out of the rod outer segment for further processing. We used fluorescence imaging to monitor retinol fluorescence and quantify the kinetics of its formation and clearance after rhodopsin bleaching in the outer segments of living isolated frog (Rana pipiens) rod photoreceptors. We independently measured the release of all-trans retinal from bleached rhodopsin in frog rod outer segment membranes and the rate of all-trans retinol removal by the lipophilic carriers interphotoreceptor retinoid binding protein (IRBP) and serum albumin. We find that the kinetics of all-trans retinol formation in frog rod outer segments after rhodopsin bleaching are to a good first approximation determined by the kinetics of all-trans retinal release from the bleached pigment. For the physiological concentrations of carriers, the rate of retinol removal from the outer segment is determined by IRBP concentration, whereas the effect of serum albumin is negligible. The results indicate the presence of a specific interaction between IRBP and the rod outer segment, probably mediated by a receptor. The effect of different concentrations of IRBP on the rate of retinol removal shows no cooperativity and has an EC50 of 40 micromol/L.     

Trans/13-cis isomerization is essential for both the photocycle and proton pumping of bacteriorhodopsin.

We studied an analogue of bacteriorhodopsin whose chromophore is based on all-trans retinal. A five-membered ring was built around the 13-14 double bond so as to prohibit trans to 13-cis isomerization. No light-induced photochemical changes were seen, other than those due to a small amount (approximately 5%) of unbleached bacteriorhodopsin remaining in the apomembrane used for regeneration. The techniques used included flash photolysis at room and liquid nitrogen temperatures and Fourier-transform infrared difference spectroscopy. When the trans-fixed pigment was incorporated into phospholipid vesicles, no evidence of light-initiated proton pumping could be found. The results indicate that trans to 13-cis isomerization is essential for the photochemical transformation and function of bacteriorhodopsin.     

Effects of light adaptation on the purple membrane structure of Halobacterium halobium.

Absorption, circular dichroism and optical rotatory dispersion of the bacteriorhodopsin containing purple membrane form Halobacterium halobium were studied in regard to the structural stability of this membrane during the photoisomerization of the retinal of the bacteriorhodopsin from the 13-cis to the all-trans configuration. The following conclusions were reached: (a) the macromolecular structure (protein-protein interaction which may result in the possible exciton interaction of the retinal pi-pi* (NV1) transition moments and protein-lipid interaction) are not significantly altered, (b) possibilities of delocalized conformation changes of the apoprotein involving secondary and/or tertiary structure can be ruled out, (c) localized secondary structure conformation changes of the apoprotein must be limited to the involvement of no more than one or two amino acid residues and localized tertiary structure conformation changes of the apoprotein must be limited to a very short segment of the protein chain containing only a few aromatic amino acid residues, and (d) the interaction between the apoprotein and retinal seems to be relatively more pronounced when the retinal is in the all-trans form than the 13-cis from and also the apoprotein seems to impose a more pronounced dissymmetric constraint on the retinal in the all-trans form than in the 13-cis form.     

All-trans/13-cis isomerization of retinal is required for phototaxis signaling by sensory rhodopsins in Halobacterium halobium.

An analogue of all-trans retinal in which all-trans/13-cis isomerization is blocked by a carbon bridge from C12 to C14 was incorporated into the apoproteins of sensory rhodopsin I (SR-I) and sensory rhodopsin II (SR-II, also called phoborhodopsin) in retinal-deficient Halobacterium halobium membranes. The "all-trans-locked" retinal analogue forms SR-I and SR-II analogue pigments with similar absorption spectra as the native pigments. Blocking isomerization prevents the formation of the long-lived intermediate of the SR-I photocycle (S373) and those of the SR-II photocycle (S-II360 and S-II530). A computerized cell tracking and motion analysis system capable of detecting 2% of native pigment activity was used for assessing motility behavior. Introduction of the locked analogue into SR-I or SR-II apoprotein in vivo did not restore phototactic responses through any of the three known photosensory systems (SR-I attractant, SR-I repellent, or SR-II repellent). We conclude that unlike the phototaxis receptor of Chlamydomonas reinhardtii, which has been reported to mediate physiological responses without specific double-bond isomerization of its retinal chromophore (Foster et al., 1989), all-trans/13-cis isomerization is essential for SR-I and SR-II phototaxis signaling.     

Comparative study on the chromophore binding sites of rod and red-sensitive cone visual pigments by use of synthetic retinal isomers and analogues

A comparative study on the chromophore (retinal) binding sites of the opsin (R-photopsin) from chicken red-sensitive cone visual pigment (iodopsin) and that scotopsin) from bovine rod pigment (rhodopsin) was made by the aid of geometric isomers of retinal (all-trans, 13-cis, 11-cis, 9-cis, and 7-cis) and retinal analogues including fluorinated (14-F, 12-F, 10-F, and 8-F) and methylated (12-methyl) 11-cis-retinals. The stereoselectivity of R-photopsin for the retinal isomers and analogues was almost identical with that of scotopsin, indicating that the shapes of the chromophore binding sites of both opsins are similar, although the former appears to be somewhat more restricted than the latter. The rates of pigment formation from R-photopsin were considerably greater than those from scotopsin. In addition, all the iodopsin isomers and analogues were more susceptible to hydroxylamine than were the rhodopsin ones. These observations suggest that the retinal binding site of iodopsin is located near the protein surface. On the basis of the spectral properties of fluorinated analogues, a polar group in the chromophore binding site of iodopsin as well as rhodopsin was estimated to be located near the hydrogen atom at the C10 position of the retinylidene chromophore. A large difference in wavelength between the absorption maxima of iodopsin and rhodopsin was significantly reduced in the 9-cis and 7-cis pigments. On the assumption that the retinylidene chromophore is anchored rigidly at the alpha-carbon of the lysine residue and loosely at the cyclohexenyl ring, each of the two isomers would have the Schiff-base nitrogen at a position altered from that of the 11-cis pigments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Met-145 is a key residue in the dark adaptation of bacteriorhodopsin homologs.

Composition of retinal isomers in three proton pumps (bacteriorhodopsin, archaerhodopsin-1, and archaerhodopsin-2) was determined by high performance liquid chromatography in their light-adapted and dark-adapted states. In the light-adapted state, more than 95% of the retinal in all three proton pumps were in the all-trans configuration. In the dark-adapted state, there were only two retinal isomers, all-trans and 13-cis, in the ratio of all-trans: 13-cis = 1:2 for bacteriorhodopsin, 1:1 for archaerhodopsin-1, and 3:1 for archaerhodopsin-2. The difference in the final isomer ratios in the dark-adapted bacteriorhodopsin and archaerhodopsin-2 was ascribed to the methionine-145 in bacteriorhodopsin. This is the only amino acid in the retinal pocket that is substituted by phenylalanine in archaerhodopsin-2. The bacteriorhodopsin point-mutated at this position to phenylalanine dramatically altered the final isomer ratio from 1:2 to 3:1 in the dark-adapted state. This point mutation also caused a 10 nm blue-shift of the adsorption spectrum, which is similar to the shift of archaerhodopsin-2 relative to the spectra of bacteriorhodopsin and archaerhodopsin-1.