Photoexcited Rhodopsin Research Articles

Kinetics of cone specific G-protein signaling in avian photoreceptor cells.

Cone photoreceptor cells of night-migratory songbirds seem to process the primary steps of two different senses, vision and magnetoreception. The molecular basis of phototransduction is a prototypical G protein-coupled receptor pathway starting with the photoexcitation of rhodopsin or cone opsin thereby activating a heterotrimeric G protein named transducin. This interaction is well understood in vertebrate rod cells, but parameter describing protein-protein interactions of cone specific proteins are rare and not available for migratory birds. European robin is a model organism for studying the orientation of birds in the earth magnetic field. Recent findings showed a link between the putative magnetoreceptor cryptochrome 4a and the cone specific G-protein of European robin. In the present work, we investigated the interaction of European robin cone specific G protein and cytoplasmic regions of long wavelength opsin. We identified the second loop in opsin connecting transmembrane regions three and four as a critical binding interface. Surface plasmon resonance studies using a synthetic peptide representing the second cytoplasmic loop and purified G protein α-subunit showed a high affinity interaction with a K D value of 21 nM. Truncation of the G protein α-subunit at the C-terminus by six amino acids slightly decreased the affinity. Our results suggest that binding of the G protein to cryptochrome can compete with the interaction of G protein and non-photoexcited long wavelength opsin. Thus, the parallel presence of two different sensory pathways in bird cone photoreceptors is reasonable under dark-adapted conditions or during illumination with short wavelengths.

Isorhodopsin: An Undervalued Visual Pigment Analog

Rhodopsin, the first visual pigment identified in the animal retina, was shown to be a photosensitive membrane protein containing covalently bound retinal in the 11-cis configuration, as a chromophore. Upon photoexcitation the chromophore isomerizes in femtoseconds to all-trans, which drives the protein into the active state. Soon thereafter, another geometric isomer—9-cis retinal—was also shown to stably incorporate into the binding pocket, generating a slightly blue-shifted photosensitive protein. This pigment, coined isorhodopsin, was less photosensitive, but could also reach the active state. However, 9-cis retinal was not detected as a chromophore in any of the many animal visual pigments studied, and isorhodopsin was passed over as an exotic and little-relevant rhodopsin analog. Consequently, few in-depth studies of its photochemistry and activation mechanism have been performed. In this review, we aim to illustrate that it is unfortunate that isorhodopsin has received little attention in the visual research and literature. Elementary differences in photoexcitation of rhodopsin and isorhodopsin have already been reported. Further in-depth studies of the photochemical properties and pathways of isorhodopsin would be quite enlightening for the initial steps in vision, as well as being beneficial for biotechnological applications of retinal proteins.

Phototransduction in retinal cones: Analysis of parameter importance.

In daylight, cone photoreceptors in the retina are responsible for the bulk of visual perception, yet compared to rods, far less is known quantitatively about their biochemistry. This is partly because it is hard to isolate and purify cone proteins. The issue is also complicated by the synergistic interaction of these parameters in producing systems biology outputs, such as photoresponse. Using a 3-D resolved, finite element model of cone outer segments, here we conducted a study of parameter significance using global sensitivity analysis, by Sobol indices, which was contextualized within the uncertainty surrounding these parameters in the available literature. The analysis showed that a subset of the parameters influencing the circulating dark current, such as the turnover rate of cGMP in the dark, may be most influential for variance with experimental flash response, while the shut-off rates of photoexcited rhodopsin and phosphodiesterase also exerted sizable effect. The activation rate of transducin by rhodopsin and the light-induced hydrolysis rate of cGMP exerted measurable effects as well but were estimated as relatively less significant. The results of this study depend on experimental ranges currently described in the literature and should be revised as these become better established. To that end, these findings may be used to prioritize parameters for measurement in future investigations.

Exploring Light and Life

Exploring Light and Life

Time-Resolved UV-Visible Studies of Rhodopsin Provide Experimental Test of Flexible Surface Model for Lipid-Protein Interactions

Time-resolved UV/Visible absorbance of the retinal chromophore of rhodopsin [1] delivers vital information about key GPCR activation steps in a membrane lipid environment [2]. Following rhodopsin photoexcitation, a minimum of four intermediates equilibrate on the millisecond-time scale [2,3]. The first equilibrium is between the protonated Schiff base (PSB) species Meta I480 and the deprotonated SB species Meta IIa and is pH independent. A second equilibrium entails spectrally silent conversion of Meta IIa to the Meta IIb substate. The final step is protonation of Glu134 of the ERY sequence in Meta IIb to yield Meta IIbH+ whose pKa characterizes the acid-base equilibrium [3]. Absorbance measurements on the microsecond-to-hundred millisecond-time scale allowed us to study effects of POPC, DOPC, or DOPC/DOPE mixtures on the first equilibrium constant K1 and on the pKa of the final equilibrium. Results were analyzed by singular-value decomposition and were fit by a linear combination of temperature-dependent basis spectra. Notably an increase in K1 was discovered due to either PE head groups or increased acyl chain unsaturation, whereas little change in pKawas evident. According to the flexible-surface model (FSM) rhodopsin becomes a sensor of negative spontaneous (intrinsic) curvature upon light activation [4]. Competition between the curvature elastic energy and the hydrophobic mismatch at the proteolipid boundary explains the influences of lipid-protein interactions [4]. Both the lipid acyl chains and polar head groups affect the Meta I-Meta II transition, revealing how protein energetics are affected by material properties of the lipid bilayer.[1] A.V. Struts et al. (2011) Nature Struct. Mol. Biol. 18, 392-394.[2] J. Epps et al. (2006) Photochem. Photobiol. 82, 1436-1441.[3] M. Mahalingam et al. (2008) PNAS 105 17795-17800.[4] A. V. Botelho et al. (2006) Biophys. J. 91, 4464-4477.

Signal-dependent Hydrolysis of Phosphatidylinositol 4,5-Bisphosphate without Activation of Phospholipase C: IMPLICATIONS ON GATING OF DROSOPHILA TRPL (TRANSIENT RECEPTOR POTENTIAL-LIKE) CHANNEL

In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P(2) in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P(2) was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P(2) is not an inhibitor of TRPL channel activation. PI(4,5)P(2) hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P(2) levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P(2) is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.

A Nonadiabatic Ab Initio Dynamics Study on Rhodopsin and Its Analog Isorhodopsin: Chemical Dynamics Reasons behind Selection of Rhodopsin by Life

Abstract The structural difference between rhodopsin and isorhodopsin is only in the cis-position of the chromophore, but the difference leads to a large discrepancy in photoisomerization period and quantum yield. The photoinduced cis–trans isomerization dynamics of the two chromophores are investigated using a Quantum Mechanics/Molecular Mechanics trajectory surface hopping scheme. Rhodopsin shows a straightforward and fast excited-state dynamics whereas the isorhodopsin dynamics in the excited state is complicated due to differences in retinal motions and space gaps formed by surrounding residues. Consequently, the isorhodopsin → bathorhodopsin reaction is slower and less efficient. Photoexcitation of rhodopsin gives bathorhodopsin only, whereas isorhodopsin yields an analog with 9,11-di-cis-retinal in addition to bathorhodopsin. These differences explain why life uses rhodopsin rather than isorhodopsin.

Rhodopsin in Nanodiscs Has Native Membrane-like Photointermediates

Time-dependent studies of membrane protein function are hindered by extensive light scattering that impedes application of fast optical absorbance methods. Detergent solubilization reduces light scattering but strongly perturbs rhodopsin activation kinetics. Nanodiscs may be a better alternative if they can be shown to be free from the serious kinetic perturbations associated with detergent solubilization. To resolve this, we monitored absorbance changes due to photointermediates formed on the microsecond to hundred millisecond time scale after excitation of bovine rhodopsin nanodiscs and compared them to photointermediates that form in hypotonically washed native membranes as well as to those that form in lauryl maltoside suspensions at 15 and 30 °C over a pH range from 6.5 to 8.7. Time-resolved difference spectra were collected from 300 to 700 nm at a series of time delays after photoexcitation and globally fit to a sum of time-decaying exponential terms, and the photointermediates present were determined from the spectral coefficients of the exponential terms. At the temperatures and pHs studied, photointermediates formed after photoexcitation of rhodopsin in nanodiscs are extremely similar to those that form in native membrane, in particular displaying the normal forward shift of the Meta I(480) ⇄ Meta II equilibrium with increased temperature and reduced pH which occurs in native membrane but which is not observed in lauryl maltoside detergent suspensions. These results were obtained using the amount of rhodopsin in nanodiscs which is required for optical experiments with rhodopsin mutants. This work demonstrates that late, physiologically important rhodopsin photointermediates can be characterized in nanodiscs, which provide the superior optical properties of detergent without perturbing the activation sequence.

Flexible Surface Model Explains Time-Resolved Uv-Visible Studies of Rhodopsin Activation in a Membrane Lipid Environment

Rhodopsin is the quintessential GPCR--the UV/Visible absorbance of its bound chromophore provides detailed time-resolved information about the GPCR activation steps [1]. At least four species equilibrate on the millisecond time scale after rhodopsin photoexcitation in a membrane environment [2]. The first millisecond-time scale equilibrium is between the protonated Schiff base (PSB) species Meta I480 and the deprotonated SB species Meta IIa and is pH independent. Subsequent equilibria involve spectrally silent transitions of Meta IIa to produce Meta IIb and protonation of Glu134 of the E(R)Y sequence in Meta IIb to give Meta IIbH+ where pKa describes the acid-base equilibrium. We used time-resolved absorbance measurements on the microsecond-to-hundred millisecond time scale to study effects of the membrane lipid environment on the first equilibrium constant, K1, and on the pKa of the final equilibrium. Reconstituted membranes of rhodopsin with POPC, DOPC, or DOPC/DOPE mixtures were studied at 30oC. Results were analyzed by singular value decomposition and globally fit to a sum of exponential terms. We discovered a striking increase in K1 due to either PE head groups or increased acyl chain unsaturation; by contrast pKa changed little. According to the flexible surface model (FSM), there is competition between the curvature elastic energy and the hydrophobic mismatch at the proteolipid boundary that explains the above influences of lipid-protein interactions [3]. The fact that both the lipid acyl chains and polar head groups affect the Meta I-Meta II transition of rhodopsin is a striking illustration of how protein energetics in membranes are governed by chemically nonspecific properties of the lipid bilayer. [1] J. Epps et al. (2006) Photochem. Photobiol.82, 1436-1441. [2] E. Zaitseva et al. (2010) JACS132, 4815-4821. [3] A.V. Botelho et al. (2006) BJ91, 4464-4477.

Enhanced Retinal Insulin Receptor-activated Neuroprotective Survival Signal in Mice Lacking the Protein-tyrosine Phosphatase-1B Gene

Protein-tyrosine phosphatase 1B (PTP1B) has been implicated in the negative regulation of insulin signaling. We previously demonstrated that light-induced tyrosine phosphorylation of the retinal insulin receptor (IR) results in the activation of phosphoinositide 3-kinase/Akt survival pathway in rod photoreceptor cells. The molecular mechanism behind light-induced activation of IR is not known. We investigated the in vivo mechanism of IR activation and found that PTP1B activity in dark-adapted retinas was significantly higher than in light-adapted retinas. We made a novel finding in this study that the light-dependent regulation of PTP1B activity is signaled through photobleaching of rhodopsin. Conditional deletion of PTP1B in rod photoreceptors by the Cre-loxP system resulted in enhanced IR signaling. Further PTP1B activity negatively regulated the neuroprotective survival signaling in the retina. One of the challenging questions in the retina research is how mutations in human rhodopsin gene slowly disable and eventually disrupt photoreceptor functions. Our studies suggest that a defect in the photobleaching of rhodopsin and mutation in rhodopsin gene enhances the activity of PTP1B, and this activated activity could down-regulate the IR survival signaling. Our studies suggest that PTP1B antagonists could be potential therapeutic agents to treat stress-induced photoreceptor degenerations and provide further evidence that rhodopsin photoexcitation may trigger signaling events alternative to the classic phototransduction.

Protonation Switches in GPCR Activation: Physiologically Significant Rhodopsin Photointermediates

Rhodopsin is a paradigm for GPCRs, yet unlike other class A members, its bound chromophore's UV/vis absorbance provides excellent time-resolved information about GPCR activation steps. At least three species equilibrate on the millisecond time scale after rhodopsin photoexcitation in a membrane lipid environment. The first equilibrium is pH-independent and involves Meta I480, the visible absorbing, protonated Schiff base (PSB) species, and Meta IIa, the UV absorbing, deprotonated SB species. The second equilibrium, involving spectrally silent proton uptake by Meta IIa to produce Meta IIb, accounts for the fact that low pH causes anomalous disappearance of the protonated SB species, Meta I480. The equilibria affect production of the G protein-activating species R∗ and are of great interest. However they must be studied promptly because inactivation steps follow, and long illumination increases secondary photolysis of photoproducts. We used time-resolved absorbance measurements of bovine rhodopsin on the microsecond-to-hundred millisecond time scale to study kinetics of lumirhodopsin decay and the effect of membrane environment on the first equilibrium constant, K1, and on the pKa of the second equilibrium. Reconstituted membranes of rhodopsin with POPC, DOPC, or a mixture of DOPC and DOPE were studied at 30°C. We also extended previous 20°C studies of the pH dependence of the equilibria in the native disk membranes, to determine how increased temperature affects lumirhodopsin decay through the purely transient 380 nm absorbing species, Meta I380, into the final equilibrium mixture. Meta I380 has recently attracted substantial interest, since time-resolved circular dichroism measurements on the microsecond timescale suggest the chromophore has a different conformation than in later 380-nm photointermediates. Our results suggest SB deprotonation precedes other activating changes in the protein. Significant details are now emerging that give new insights into rhodopsin activation and complement FTIR and spin-label approaches.

Configuration analysis of the electronic excitation in photobiological receptors

According to configuration analysis of the electronic state wave functions of chlorophyll a, peripheral CC(3b,4b) and isocyclic CO groups are not locally excited to any significant extent. This contrasts with the pyrone CC bond of coumarins where a substantial localization of the triplet excitation as well as photoreactions occur. The lowest excited states (1QQ and 3QQ) of chlorophyll a and bacteriochlorophyll a are also similar in correlating their state wave functions, although the ground states of these molecules are substantially different. The 11-CC bond of 11-cis-retinal protonated Schiff's bases shows no tendency for a localized excitation. Thus the primary photoexcitation of rhodopsin does not involve a preferential excitation at the 11-CC bond. The fluorescence characteristics of firefly luciferins have also been analyzed by the configuration analysis method. The most significant result is that the fluorescent states of luciferin and oxyluciferin monoanion possess a strong charge transfer character from hydroxybenzothiazole to thiazoline, in agreement with experimental observations.

Growth Factor Receptor-Bound Protein 14 Undergoes Light-Dependent Intracellular Translocation in Rod Photoreceptors: Functional Role in Retinal Insulin Receptor Activation

Growth factor receptor-bound protein 14 (Grb14) is involved in growth factor receptor tyrosine kinase signaling. Here we report that light causes a major redistribution of Grb14 among the individual subcellular compartments of the retinal rod photoreceptor. Grb14 is localized predominantly to the inner segment, nuclear layer, and synapse in dark-adapted rods, whereas in the light-adapted rods, Grb14 redistributed throughout the entire cell, including the outer segment. The translocation of Grb14 requires photoactivation of rhodopsin, but not signaling through the phototransduction cascade, and is not based on direct Grb14-rhodopsin interactions. We previously hypothesized that Grb14 protects light-dependent insulin receptor (IR) activation in rod photoreceptors against dephosphorylation by protein tyrosine phosphatase 1B. Consistent with this hypothesis, we failed to observe light-dependent IR activation in Grb14(-/-) mouse retinas. Our studies suggest that Grb14 translocates to photoreceptor outer segments after photobleaching of rhodopsin and protects IR phosphorylation in rod photoreceptor cells. These results demonstrate that Grb14 can undergo subcellular redistribution upon illumination and suggest that rhodopsin photoexcitation may trigger signaling events alternative to the classical transducin activation.

Real-Time Dynamics Changes of Helix 8 upon Phosphorylation of Rhodopsin and Arrestin Binding

The G-protein coupled receptor (GPCR) rhodopsin transduces light signals in photoreceptor cells. For a rapid recovery of the receptor, efficient signal termination is mandatory. On the molecular level, this termination process starts with the phosphorylation of serines and/or threonines in the C-terminus of rhodopsin by rhodopsin kinase. Subsequently, the phosphorylated photoexcited rhodopsin is recognized by visual arrestin, which binds to the cytoplasmic loops of the receptor.In this work we focus on the real-time dynamics of helix 8, a putative cytoplasmic surface switch region in class-I GPCRs. Using multidimensional time-correlated single photon counting [1] and site-specific fluorescence labelling [2], we measured the time-resolved anisotropy changes of phosphorylated and photoactivated phosphorylated rhodopsin upon interaction with arrestin. Distinct real-time dynamics changes of helix 8 were observed after phosphosphorylation and interaction with arrestin. From real-time dynamics studies of helix 8 in rod outer segment membranes we concluded that a reduced mobility of helix 8 is a prerequisite for rhodopsin activation [2]. FTIR-Fluorescence cross-correlation measurements indicate further that changes in the environment of helix 8, as measured with a fluorescent reporter group, may correlate with a partial loss of secondary structure upon Metarhodopsin II formation [3]. The observed mobility changes after phosphorylation highlight the importance of helix 8 as a surface element, which is involved in activation, molecular recognition, and deactivation of the receptor.[1] Kim T.-Y., Winkler K., and U. Alexiev (2007). Photochemistry and Photobiology 83, 378-384[2] Alexiev, U., I. Rimke, and T. Pohlmann (2003). J. Mol. Biol. 328, 705-719[3] Lehmann N., Alexiev U., and Fahmy K. J (2007). Mol. Biol. 366, 1129-1141

Mouse Cones Require an Arrestin for Normal Inactivation of Phototransduction

Arrestins are proteins that arrest the activity of G protein-coupled receptors (GPCRs). While it is well established that normal inactivation of photoexcited rhodopsin, the GPCR of rod phototransduction, requires arrestin (Arr1), it has been controversial whether the same requirement holds for cone opsin inactivation. Mouse cone photoreceptors express two distinct visual arrestins: Arr1 and Arr4. By means of recordings from cones of mice with one or both arrestins knocked out, this investigation establishes that a visual arrestin is required for normal cone inactivation. Arrestin-independent inactivation is 70-fold more rapid in cones than in rods, however. Dual arrestin expression in cones could be a holdover from ancient genome duplication events that led to multiple isoforms of arrestin, allowing evolutionary specialization of one form while the other maintains the basic function.

DAG Lipase Activity Is Necessary for TRP Channel Regulation in Drosophila Photoreceptors

In Drosophila, a phospholipase C-mediated signaling cascade links photoexcitation of rhodopsin to the opening of the TRP/TRPL channels. A lipid product of the cascade, diacylglycerol (DAG) and its metabolite(s), polyunsaturated fatty acids (PUFAs), have both been proposed as potential excitatory messengers. A crucial enzyme in the understanding of this process is likely to be DAG lipase (DAGL). However, DAGLs that might fulfill this role have not been previously identified in any organism. In this work, the Drosophila DAGL gene, inaE, has been identified from mutants that are defective in photoreceptor responses to light. The inaE-encoded protein isoforms show high sequence similarity to known mammalian DAG lipases, exhibit DAG lipase activity in vitro, and are highly expressed in photoreceptors. Analyses of norpA inaE double mutants and severe inaE mutants show that normal DAGL activity is required for the generation of physiologically meaningful photoreceptor responses.

Photons, femtoseconds and dipolar interactions: a molecular picture of the primary events in vision.

The first 200 femtoseconds in the life of a photoexcited rhodopsin molecule are extremely important for the development of visual sensation. Immediately upon excitation, a dramatic change in the charge distribution of the cationic 11-cis-retinal protonated Schiff base chromophore occurs that is quantitated by the change in electronic dipole moment of approximately 15 Debye. The opsin protein tunes the absorption maximum of the pigment to the blue or to the red enabling colour vision by placing dipolar rather than charged residues in the chromophore binding site to differentially stabilize either the ground or the excited state charge distribution. Resonance Raman intensity analysis reveals that the 11-cis-retinal chromophore then distorts violently about the C11 = C12 double bond, reaching torsional angles of approximately 70 degrees in only 30 fs. This rapid torsional distortion is driven by the non-bonded interaction between the 13-methyl group and the 10-hydrogen that is unique to the 11-cis configuration of the chromophore. The excited state depopulates in approximately 50 fs through a rapid and vibrationally coherent transition to the ground electronic state manifold with relaxation to the formally trans photoproduct complete in only 200 fs. This unusually fast and efficient isomerization process establishes a new paradigm for condensed phase photochemical reactions.

Regulation of Rod Phototransduction Machinery by Ciliary Neurotrophic Factor

Ciliary neurotrophic factor (CNTF) promotes photoreceptor survival but also suppresses electroretinogram (ERG) responses. This has caused concerns about whether CNTF is detrimental to the function of photoreceptors because it is considered to be a potential treatment for retinal degenerative disorders. Here we report that the suppression of ERG responses is attributable to negative regulation of the phototransduction machinery in rod photoreceptors. Intravitreal injection of recombinant human CNTF protein in rat results in a series of biochemical and morphological changes in rod photoreceptors. CNTF induces a decrease in rhodopsin expression and an increase in arrestin level. Morphologically, CNTF treatment causes a shortening of rod outer segments (ROS). All of these changes are fully reversible. The lower rhodopsin level and shortened ROS reduce the photon catch of rods. Less rhodopsin and more arrestin dramatically increase the arrestin-to-rhodopsin ratio so that more arrestin molecules are available to quench the photoexcited rhodopsin. The overall effect of CNTF is to negatively regulate the phototransduction machinery, which reduces the photoresponsiveness of rods, resulting in lower ERG amplitude at a given intensity of light stimulus. The CNTF-induced changes in rods are similar to those in light-induced photoreceptor plasticity. Whether CNTF-induced changes in rods are through the same mechanism that mediates light-induced photoreceptor plasticity remains to be answered.

Lumi I → Lumi II: The Last Detergent Independent Process in Rhodopsin Photoexcitation

Time-resolved absorbance difference spectra were collected at delays from 1 to 128 μs after photolysis of membrane and detergent suspensions of rhodopsin at 20°C. Fitting both sets of data with two exponential decays plus a constant showed a similar fast process (lifetime 11 μs in membrane, 12 μs in 5% dodecyl maltoside) with a small but similar spectral change. This demonstrates that the Lumi I → Lumi II process, previously characterized in detergent suspensions, has similar properties in membrane without significant effect of detergent. The slower exponential process detected in the data is quite different in membrane compared to detergent solubilized samples, showing that the pronounced effect of detergent on the later rhodopsin photointermediates begins fairly abruptly near 20 μs. Besides affecting the late processes, the data collected here shows that detergent induces a small blue shift in the 1 μs difference spectrum (the Lumi I minus rhodopsin difference spectrum). The blue shift is similar to one induced by chloride ion in the E181Q rhodopsin mutant and may indicate that the ionization state of Glu181 in rhodopsin is affected by detergent.

Lumi I → Lumi II: The Last Detergent Independent Process in Rhodopsin Photoexcitation†

Time-resolved absorbance difference spectra were collected at delays from 1 to 128 micros after photolysis of membrane and detergent suspensions of rhodopsin at 20 degrees C. Fitting both sets of data with two exponential decays plus a constant showed a similar fast process (lifetime 11 micros in membrane, 12 micros in 5% dodecyl maltoside) with a small but similar spectral change. This demonstrates that the Lumi I - Lumi II process, previously characterized in detergent suspensions, has similar properties in membrane without significant effect of detergent. The slower exponential process detected in the data is quite different in membrane compared to detergent solubilized samples, showing that the pronounced effect of detergent on the later rhodopsin photointermediates begins fairly abruptly near 20 micros. Besides affecting the late processes, the data collected here shows that detergent induces a small blue shift in the 1 micros difference spectrum (the Lumi I minus rhodopsin difference spectrum). The blue shift is similar to one induced by chloride ion in the E181Q rhodopsin mutant and may indicate that the ionization state of Glu181 in rhodopsin is affected by detergent.