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Cone photoreceptors of the vertebrate retina terminate their response to light much faster than rod photoreceptors. However, the molecular mechanisms underlying this rapid response termination in cones are poorly understood. The experiments presented here tested two related hypotheses: first, that the rapid decay rate of metarhodopsin (Meta) II in red-sensitive cones depends on interactions between the 9-methyl group of retinal and the opsin part of the pigment molecule, and second, that rapid Meta II decay is critical for rapid recovery from saturation of red-sensitive cones after exposure to bright light. Microspectrophotometric measurements of pigment photolysis, microfluorometric measurements of retinol production, and single-cell electrophysiological recordings of flash responses of salamander cones were performed to test these hypotheses. In all cases, cones were bleached and their visual pigment was regenerated with either 11-cis retinal or with 11-cis 9-demethyl retinal, an analogue of retinal lacking the 9-methyl group. Meta II decay was four to five times slower and subsequent retinol production was three to four times slower in red-sensitive cones lacking the 9-methyl group of retinal. This was accompanied by a significant slowing of the recovery from saturation in cones lacking the 9-methyl group after exposure to bright (>0.1% visual pigment photoactivated) but not dim light. A mathematical model of the turn-off process of phototransduction revealed that the slower recovery of photoresponse can be explained by slower Meta decay of 9-demethyl visual pigment. These results demonstrate that the 9-methyl group of retinal is required for steric chromophore--opsin interactions that favor both the rapid decay of Meta II and the rapid response recovery after exposure to bright light in red-sensitive cones.

Our ability to see in bright light depends critically on the rapid rate at which cone photoreceptors detect and adapt to changes in illumination. This is achieved, in part, by their rapid response termination. In this study, we investigate the hypothesis that this rapid termination of the response in red cones is dependent on interactions between the 9-methyl group of retinal and red cone opsin, which are required for timely metarhodopsin (Meta) II decay. We used single-cell electrical recordings of flash responses to assess the kinetics of response termination and to calculate guanylyl cyclase (GC) rates in salamander red cones containing native visual pigment as well as visual pigment regenerated with 11-cis 9-demethyl retinal, an analogue of retinal in which the 9-methyl group is missing. After exposure to bright light that photoactivated more than ~0.2% of the pigment, red cones containing the analogue pigment had a slower recovery of both flash response amplitudes and GC rates (up to 10 times slower at high bleaches) than red cones containing 11-cis retinal. This finding is consistent with previously published biochemical data demonstrating that red cone opsin regenerated in vitro with 11-cis 9-demethyl retinal exhibited prolonged activation as a result of slowed Meta II decay. Our results suggest that two different mechanisms regulate the recovery of responsiveness in red cones after exposure to light. We propose a model in which the response recovery in red cones can be regulated (particularly at high light intensities) by the Meta II decay rate if that rate has been inhibited. In red cones, the interaction of the 9-methyl group of retinal with opsin promotes efficient Meta II decay and, thus, the rapid rate of recovery.

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50-70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10-40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.

Rod vision is initiated when 11-cis-retinal, bound within rhodopsin, absorbs a photon and isomerizes to all-transretinal (ATR). This triggers an enzyme cascade that lowers cGMP, thereby closing cyclic nucleotide-gated (CNG) channels. ATR then dissociates from rhodopsin, with bright light releasing millimolar levels of ATR. We have recently shown that ATR is a potent closed-state inhibitor of the rod CNG channel, and that it requires access to the cytosolic face of the channel (McCabe, S.L., D.M. Pelosi, M. Tetreault, A. Miri, W. Nguitragool, P. Kovithvathanaphong, R. Mahajan, and A.L. Zimmerman. 2004. J. Gen. Physiol. 123:521-531). However, the details of the interaction between the channel and ATR have not been resolved. Here, we explore the nature of this interaction by taking advantage of specific retinoids and retinoid analogues, namely, [beta]-ionone, all-trans-C15 aldehyde, all-trans-C17 aldehyde, all-trans-C22 aldehyde, all-trans-retinol, all-trans-retinoic acid, and all-trans-retinylidene-n-butylamine. These retinoids differ in polyene chain length, chemical functionality, and charge. Results obtained from patch clamp and NMR studies have allowed us to better define the characteristics of the site of retinoid--channel interaction. We propose that the cytoplasmic face of the channel contains a retinoid binding site. This binding site likely contains a hydrophobic region that allows the ionone ring and polyene tail to sit in an optimal position to promote interaction of the terminal functional group with residues ~15 [Angstrom] away from the ionone ring. Based on our functional data with retinoids possessing either a positive or a negative charge, we speculate that these amino acid residues may be polar and/or aromatic.

A triple mutant of the tiger salamander UV cone pigment appears to be trapped in an open conformation that is metarhodopsin-II-like. The triple mutant represents the first example of a dark-active cone pigment and constitutively active cone opsin.

The visual cycle comprises a sequence of reactions that regenerate the visual pigment in photoreceptors during dark adaptation, starting with the reduction of all-trans retinal to all-trans retinol and its clearance from photoreceptors. We have followed the reduction of retinal and clearance of retinol within bleached outer segments of red rocks isolated from salamander retina by measuring its intrinsic fluorescence, Following exposure to a bright light (bleach), increasing fluorescence intensity was observed to propagate along the outer segments in a direction front the proximal region adjacent to the inner segment toward the distal tip. Peak retinol fluorescence was achieved after ~30 min, after which it declined very slowly. Clearance of retinol fluorescence is considerably accelerated by the presence of the exogenous lipophilic substances IRBP (interphotoreceptor retinoid binding protein) and serum albumin. We have used simultaneous fluorometric and electrophysiological measurements to compare the rate of reduction of all-trans retinal to all-trans retinol to the rate of recovery of flash response amplitude in these cells in the presence and absence of IRBP. We find that flash response recovery in rods is modestly accelerated in the presence of extracellular IRBP. These results suggest such substances may participate in the clear, race of retinoids from rod photoreceptors, and that this clearance, at least in rods, may facilitate dark adaptation by accelerating the clearance of photoproducts of bleaching. KEY WORDS: retinol * vitamin A * photoreceptor * visual cycle * IRBP

Post-translational modification in the fiber cell of ocular lenses and the intralenticular distribution of the modified forms of Aquaporin 0 (AQP0), most abundant protein in the human lenses, is investigated. Findings indicated that the spatial distribution of C-terminally modified AQP0 within the lens cause an increase in truncation and racemization/isomerization with fiber cell age.

Results demonstrate that the hydroxyl group of Tyr83 is involved in the efficient functioning and stability of bacteriorhodopsin. Mutational studies indicate that Tyr83 mediates proton release and proton transport from Asp85 to proton release complex.

Research has been conducted on the interhelical loop I of rhodopsin. The site-specific cleavage on this loop has been investigated.

The interaction of the R134 and E194 groups of bacteriorhodopsin affects the release of a proton from the D85 group following light stimulation. The rate of this deprotonation controls the transition between the O and bR states.

Research was conducted to examine the influence of C19 and C20 methyl group removals on isomerization of the retinols in retinal pigment epithelial cells using all-trans-9-desmethylretinol and all-trans-13-desmethylretinol. Results provide evidence that the optimal isomerization condition necessitates the presence, not only apo-rCRALBP, but also BSA in vitro. The possibility that the C19 methyl group is essential for isomerization is indicated.

The pH dependence of the O photointermediate of the bacteriorhodopsin (bR) photocycle was examined using wild-type (WT) pigment and mutants of Glu-194, Glu-204, and residues of the proton release group (PRG) in bR. Results showed that in WT, the rate constant of O to bR transition declined 30-fold upon a decrease of the pH from 6 to 3 with a pKa of around 4.3. D2O was also demonstrated to slow the rise and decay of the O intermediate in the WT at PH 3.5 by a factor of 5.5. Further analysis indicated that the rate of the O to bR transition at low pH is guided by the deprotonation of the PRG.

Analysis of the absorption spectra of constructed and expressed E194C mutant in Halobacterium salinarium was used in establishing the influence of glutamate-194 cysteine mutations on fast light-induced proton release in bacteriorhodopsin. Results have shown that the substitution of glu-194 with cysteine completely suppresses fast light-induced proton release at neutral pH, mimicking the effect of E204Q mutation. The pH dependence of the rate constant in E194C reveals that the coupling is reduced.

The effects of proton uptake and release in the photocycle of the bacteriorhodopsin mutant E204Q were analyzed by utilizing flash-induced absorbance, decay, and pH dependence studies on transformed E204Q mutant purple membrane. It was found that the photocycling time of mutant E204Q increased following substitution by glutamine altering the proton release uptake event from M to O intermediate. Furthermore, deprotonated negatively charged form of azides and weak acids catalyze deprotonation of D85 by acting as proton recipients.