Fast electrical potentials arising from activation of metarhodopsin in the fly

The cellular origin and properties of fast electrical potentials arising from activation of Calliphora photopigment were investigated. It was found by intracellular recordings that only the corneal-negative M1 phase of fly M potential arises in the photoreceptors' membrane. This M1 phase has all the accepted characteristics of an early receptor potential (ERP). It has no detectable latency, it survives fixation with glutaraldehyde, it is linear with light intensity below pigment saturation, and it is linear with the amount of metarhodopsin activated by light. The Calliphora ERP was found, however, to be exceptional because activation of rhodopsin, which causes the formation of metarhodopsin in 125 microsecond (25 degrees C), was not manifested in the ERP. Also, the extracellularly recorded ERP was not proportional to the rate of photopigment conversion. The corneal-positive M2 phase of the M potential was found to arise from second-order lamina neurons (L neurons). Intracellular recordings from these cells showed a fast hyperpolarizing potential, which preceded the normal hyperpolarizing transient of these cells. This fast potential appeared only when metarhodopsin was activated by a strong flash. The data indicate that the intracellularly recorded positive ERP, which arises from activation of metarhodoposin, elicits a hyperpolarizing fast potential in the second-order neuron. This potential is most likely the source of the corneal-positive M potential.     

Non-local interactions between light induced processes inCalliphora photoreceptors

Summary The prolonged depolarizing afterpotential (PDA) is a phenomenon which is tightly linked to visual pigment conversion. In order to determine whether processes underlying PDA induction and depression can spread in space, the PDA was recorded intracellularly in white-eyedCalliphora R1-6 photoreceptors and used to examine interactions between processes induced by activating statistically different photopigment molecules (Figs. 3–6). It was found that a PDA induced by converting some fraction of rhodopsin (R) molecules forward into the metarhodopsin (M) state can be completely depressed by equal or smaller amounts of pigment conversion, backward from metarhodopsin to rhodopsin even when largely different sets of pigment molecules were shifted in the respective directions, in agreement with previous experiments conducted on the barnacle. The characteristics of the afterpotentials obtained following the cessation of strong blue and green light stimuli which did not cause a net pigment conversion was examined (Figs. 7, 8). It was found that these afterpotentials, obtained when nonet R to M conversion took place, could not be depressed by an opposite net large M to R pigment conversion. Accordingly we propose to restrict the term PDA to an afterpotential which can be depressed by a net M to R pigment conversion. It is concluded: (a) that some processes underlying PDA induction and depression inCalliphora must interact at a distance which extends at least to the nearest neighboring pigment molecule, and (b) that inCalliphora photoreceptors net pigment conversion is required in order to induce and depress a PDA.Abbreviations R rhodopsin - M metarhodopsin - R to M rhodopsin to metarhodopsin pigment conversion - M to R metarhodopsin to rhodopsin pigment conversion - PDA prolonged depolarizing afterpotential - ERG electroretinogram - M potential metarhodopsin potential - ERP early receptor potential     

Spatial properties of the prolonged depolarizing afterpotential in barnacle photoreceptors. I. The induction process

In invertebrate photoreceptors, when the light stimulus results in substantial net transfer of the visual pigment from the rhodopsin (R) to the metarhodopsin (M) state, the ordinary late receptor potential (LRP) is followed by a prolonged depolarizing afterpotential (PDA). The dependence of the amplitude of the PDA on the amount of pigment conversion is strongly supralinear, and the PDA duration also depends on this amount. These observations indicate an interaction among the elements of the PDA induction process and also make possible a test of the range of this interaction. The test consists of a comparison of the PDA after localized pigment conversion, obtained by strong spot illumination, to that after weaker diffuse illumination converting a comparable total amount of pigment. The experiment was performed on the barnacle lateral eye. The effective spot size was measured by the early receptor potential (ERP), in seawater saturated with CO2, which considerably reduced the electrical coupling between the photoreceptors. The ERP was also used to determine whether there is diffusion of R molecules into the illuminated spot. The spot illumination induced a PDA with small amplitude and long duration, while no detectable PDA was induced by the diffuse light. This indicates that the range of the PDA interaction is much smaller than the entire cell. In addition, the ERP results showed that there was no detectable diffusion of R molecules into the illuminated spot area over 30 min. This measurement, with a calculated correction for the microvillar geometry of the photoreceptor, enabled us to put an upper limit on the diffusion coefficient of the pigment molecules in the inact, unfixed barnacle photoreceptor of D less than 6 X 10(-9) cm2 s-1.     

Fast Electrical Potential from a Long-Lived,Long-Wavelength Photoproduct of Fly Visual Pigment

A rapid electrical potential, which we have named the M-potential, can be obtained from the Drosophila eye using a high energy flash stimulus. The potential can be elicited from the normal fly, but it is especially prominent in the mutant norp A^P12 (a phototransduction mutant), particularly if the eye color pigments are genetically removed from the eye. Several lines of evidence suggest that the M-potential arises from photoexcitation of long-lived metarhodopsin. Photoexcitation of rhodopsin does not produce a comparable potential. The spectral sensitivity of the M-potential peaks at about 575 nm. The M-potential pigment (metarhodopsin) can be shown to photoconvert back and forth with a "silent pigment(s)" absorbing maximally at about 485 nm. The silent pigment presumably is rhodopsin. These results support the recent spectrophotometric findings that dipteran metarhodopsin absorbs at much longer wavelengths than rhodopsin. The M-potential probably is related to the photoproduct component of the early receptor potential (ERP). Two major differences between the M-potential and the classical ERP are: (a) Drosophila rhodopsin does not produce a rapid photoresponse, and (b) an anesthetized or freshly sacrificed animal does not yield the M-potential. As in the case of the ERP, the M-potential appears to be a response associated with a particular state of the fly visual pigment. Therefore, it should be useful in in vivo investigations of the fly visual pigment, about which little is known.     

Analysis of the rhodopsin cycle in limulus ventral photoreceptors using the early receptor potential

The early receptor potential (ERP) was recorded intracellularly from Limulus ventral photoreceptors. The ERP in cells dissected under red light was altered by exhaustive illumination. No recovery to the original wafeform was observed, even after 1 h in the dark. The ERP waveform could be further altered by chromatic adaptation or by changes in pH. The results indicate that at pH 7.8 there are two interconvertible pigment states with only slightly different lambdamax, whereas at pH 9.6 there are two interconvertible states with very different lambdamax. Under all conditions studied the ERPs were almost identical with those previously obtained in squid retinas. This strongly suggests that light converts Limulus rhodopsin to a stable photoequilibrium mixture of rhodopsin to a stable photoequilibrium mixture of rhodopsin and metarhodopsin and that, as in squid, the lambdamax of metarhodopsin depends on pH. This conversion at pH 7.8 is associated with a small (0.7 log unit) decrease in the maximum sensitivity of the late receptor potential. Thus the component of adaptation linked to changes in rhodopsin concentration is unimportant in comparison to the "neural" component.     

The contribution of a sensitizing pigment to the photosensitivity spectra of fly rhodopsin and metarhodopsin.

Most of the photoreceptors of the fly compound eye have high sensitivity in the ultraviolet (UV) as well as in the visible spectral range. This UV sensitivity arises from a photostable pigment that acts as a sensitizer for rhodopsin. Because the sensitizing pigment cannot be bleached, the classical determination of the photosensitivity spectrum from measurements of the difference spectrum of the pigment cannot be applied. We therefore used a new method to determine the photosensitivity spectra of rhodopsin and metarhodopsin in the UV spectral range. The method is based on the fact that the invertebrate visual pigment is a bistable one, in which rhodopsin and metarhodopsin are photointerconvertible. The pigment changes were measured by a fast electrical potential, called the M potential, which arises from activation of metarhodopsin. We first established the use of the M potential as a reliable measure of the visual pigment changes in the fly. We then calculated the photosensitivity spectrum of rhodopsin and metarhodopsin by using two kinds of experimentally measured spectra: the relaxation and the photoequilibrium spectra. The relaxation spectrum represents the wavelength dependence of the rate of approach of the pigment molecules to photoequilibrium. This spectrum is the weighted sum of the photosensitivity spectra of rhodopsin and metarhodopsin. The photoequilibrium spectrum measures the fraction of metarhodopsin (or rhodopsin) in photoequilibrium which is reached in the steady state for application of various wavelengths of light. By using this method we found that, although the photosensitivity spectra of rhodopsin and metarhodopsin are very different in the visible, they show strict coincidence in the UV region. This observation indicates that the photostable pigment acts as a sensitizer for both rhodopsin as well as metarhodopsin.     

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     

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.     

Upper limit on translational diffusion of visual pigment in intact unfixed barnacle photoreceptors

Translational diffusion of pigment molecules in the disc membranes of amphibian rod outer segments is in the range of 10 /10 s. Recently, Goldsmith and Wehner set an upper limit of 10 /20 min to the diffusion in isolated formaldehyde-fixed rhabdoms of crayfish. We have now used the early receptor potential (ERP) to study the diffusion in intact, unfixed barnacle photoreceptors. The ERP from a cell fully adapted to blue light (most of the pigment in the rhodopsin state) was changed by 8–22% of its maximum change when the pigment in a 30 m spot was (almost) completely shifted to the metarhodopsin state by red laser adaptation. Further red illumination of the same spot 30 min later produced only a limited further change in the ERP (attributable to light scatter), showing that R had not migrated into the spot. It is concluded that the visual pigment diffuses by less than 30 /30 min.Based on material presented at the European Neurosciences Meeting, Florence, September 1978     

Light-induced pigment granule migration in the retinular cells of Drosophila melanogaster. Comparison of wild type with ERG-defective mutants

The dependence of pigment granule migration (PGM) upon the receptor potential was examined using several strains of electroretinogram (ERG)- defective mutants of Drosophila melanogaster. The mutants that have a defective lamina component but a normal receptor component of the ERG (no on-transient A [nonA] and tan) exhibited normal pigment granule migration. The mutants that have very small or no receptor potentials (certain no receptor potential A [norpA] alleles), on the other hand, exhibited no PGM. In the case of the temperature-sensitive norpA mutant, norpAH52, normal PGM was present at 17 degrees but not at 32 degrees C or above, corresponding to its electrophysiological phenotype. In the transient receptor potential (trp) mutant, whose receptor potential decays to the baseline within a few seconds during a sustained light stimulus, the pigment granules initially moved close to the rhabdomere when light was turned on but moved away after about 5 s during a sustained light stimulus. All these results lend strong support to the notion that PGM is initiated by a light-evoked depolarization of the receptor membrane, i.e., the receptor potential. However, under certain experimental conditions, the receptor potentials failed to induce PGM in the trp mutant. The depolarization of the receptor, thus, appears to be closely associated with PGM but is not a sufficient condition for PGM.     

The contribution of pigment transitions to sensitivity changes in the barnacle photoreceptor and the correlation with the prolonged depolarizing afterpotential

A conditioning light can cause a decrease (adaptation) or an increase (facilitation) in the sensitivity of barnacle photoreceptors, as measured by the amplitude of the late receptor potential (LRP). We show that a net transfer of visual pigment from the rhodopsin (R) to the metarhodopsin (M) state induces a large facilitation whereas the reverse transfer results in a much smaller facilitation or even an adaptation. These effects were not due to the response to the conditioning light but to the pigment reactions. When the conditioning light did not alter the pigment population (i.e., M M, R R) it was followed by an intermediate degree of facilitation. These conclusions are correct for cells which have relatively low sensitivity. In sensitive cells, all pigment transitions produce adaptation.LRP facilitation and the prolonged depolarizing afterpotential (PDA) show several common characteristics with respect to pigment transitions: 1.Their magnitude increases with the amount of pigment transferred from R to M. 2. Both are depressed by the M R transition. 3. Their production is impeded by the M R transition. 4. The PDA itself is facilitated by the R M transition and this facilitation decays with a time course comparable to that of LRP facilitation. These results suggest that there may be an underlying process common to LRP facilitation and PDA.     

Transduction in photoreceptors with bistable pigments: Intermediate processes

The prolonged depolarizing after potential (PDA) in the R1–6 receptors of the fly was used to isolate intermediate processes in phototransduction which are not manifested directly in the voltage response. It is first demonstrated that a pigment shift by light from metarhodopsin to rhodopsin in four species of the flies: Drosophila, Calliphora, Chrysomya and Musca induces an independent antagonistic process to the PDA, which is manifested in a strong inhibitory effect on PDA induction and is called the anti-PDA.By using mutants of Drosophila the existence of processes underlying the PDA were examined. The norpA ^H52and the trp mutant were used in which the voltage response of the photoreceptors could be reversibly abolished by elavated temperature and long intense light respectively. It is shown that the excitatory process underlying the PDA could be induced and depressed in conditions that block the voltage response of the photoreceptors, thus indicating the existance of intermediate processes which link the pigment activation by light to the PDA voltage response.Based on material presented at the European Neurosciences Meeting, Florence, September 1978     

Afterpotentials in dronefly retinula cells

Summary The wavelength dependence of the afterpotentials following a bright illumination was studied in single photoreceptor cells of the droneflyEristalis. Cells with only a spectral sensitivity peak in the blue were selected. As previously demonstrated, these cells contain a rhodopsin absorbing maximally at about 450–460 nm, which upon photoconversion transforms into a metarhodopsin absorbing maximally at about 550 nm (Tsukahara and Horridge, 1977).With the visual pigment initially all in the rhodopsin form, a high rate of visual pigment conversion results in an afterhyperpolarization (AHP) when the fraction of metarhodopsin remains negligible after illumination as occurs at longer wavelengths if the intensity is high. Intensive illumination at short wavelengths is followed by a prolonged depolarizing afterpotential (PDA). The magnitude of the PDA peaks at low intensities at about 450–460 nm, corresponding to the peak of the cell's spectral sensitivity (i.e. the rhodopsin peak). With increasing intensity of illumination, however, the peak shifts progressively towards 430 nm, which corresponds to the photoequilibrium with maximum metarhodopsin that can be established by monochromatic light. From this result, it is inferred that the PDA is related to the induced fall in the rhodopsin fraction. The PDA can be abolished, or knocked down, by a long-wavelength flash which reconverts remaining metarhodopsin into rhodopsin. Therefore the decline of the PDA is restrained by the existing amount of metarhodopsin. Possible theories of afterpotentials are discussed.     

Müller glia endfeet,a basal lamina and the polarity of retinal layers form properly in vitro only in the presence of marginal pigmented epithelium

Summary Dissociated embryonic chicken retinal cells regenerate in rotary culture into cellular spheres that consist of subareas expressing all three nuclear layers in an inside-out sequence (rosetted vitroretinae). However, when pigmented cells from the eye margin (peripheral retinal pigment epithelium) are added to the system, the sequence of layers is identical with that of an in-situ retina (laminar vitroretinae). In order to elucidate further the lamina-stabilizing effect exerted by the retinal pigment epithelium, we have compared both systems, laying particular emphasis on the ultrastructure of the basal lamina and of Müller glia processes. Ultrastructurally, in both systems, an outer limiting membrane, inner segments of photoreceptors and the segregation of cell bodies into three cell layers develop properly. Synapses are detectable in a premature state, although only in the inner plexiform layer of laminar vitroretinae. Although present in both systems, radial processes of juvenile Müller glia cells are properly fixed at their endfeet only in laminar vitroretinae, since a basal lamina is only expressed here. Large amounts of laminin are detected immunohistochemically within the retinal pigment epithelium and along a basal stalk that reaches inside the laminar vitroretinae. We conclude that the peripheral retinal pigment epithelium is essential for the expression of a basal lamina in vitro. Moreover, the basal lamina may be responsible both for stabilizing the correct polarity of retinal layers and for the final differentiation of the Müller cells.     

Photoreceptor responses of fruitflies with normal and reduced arrestin content studied by simultaneous measurements of visual pigment fluorescence and ERG

We have simultaneously measured the electroretinogram (ERG) and the metarhodopsin content via fluorescence in white-eyed, wild-type Drosophila and the arrestin2 hypomorphic mutant (w ⁻ ;arr2 ³ ) at a range of stimulus wavelengths and intensities. Photoreceptor response amplitude and termination (transition between full repolarization and prolonged depolarizing afterpotential, PDA) were related to visual pigment conversions and arrestin concentration. The data were implemented in a kinetic model of the rhodopsin–arrestin cycle, allowing us to estimate the active metarhodopsin concentration as a function of effective light intensity and arrestin concentration. Arrestin reduction in the mutant modestly increased the light sensitivity and decreased the photoreceptor dynamic range. Compared to the wild type, in the mutant the transition between full repolarization and PDA occurred at a lower metarhodopsin fraction and was more abrupt. We developed a steady-state stochastic model to interpret the dependence of the PDA on effective light intensity and arrestin content and to help deduce the arrestin to rhodopsin ratio from the sensitivity and PDA data. The feasibility of different experimental methods for the estimation of arrestin content from ERG and PDA is discussed.     

Responses of crayfish photoreceptor cells following intense light adaptation

Summary After intense orange adapting exposures that convert 80% of the rhodopsin in the eye to metarhodopsin, rhabdoms become covered with accessory pigment and appear to lose some microvillar order. Only after a delay of hours or even days is the metarhodopsin replaced by rhodopsin (Cronin and Goldsmith 1984). After 24 h of dark adaptation, when there has been little recovery of visual pigment, the photoreceptor cells have normal resting potentials and input resistances, and the reversal potential of the light response is 10–15 mV (inside positive), unchanged from controls. The log V vs log I curve is shifted about 0.6 log units to the right on the energy axis, quantitatively consistent with the decrease in the probability of quantum catch expected from the lowered concentration of rhodopsin in the rhabdoms. Furthermore, at 24 h the photoreceptors exhibit a broader spectral sensitivity than controls, which is also expected from accumulations of metarhodopsin in the rhabdoms. In three other respects, however, the transduction process appears to be light adapted: (i) The voltage responses are more phasic than those of control photoreceptors. (ii) The relatively larger effect (compared to controls) of low extracellular Ca⁺⁺ (1 mmol/1 EGTA) in potentiating the photoresponses suggests that the photoreceptors may have elevated levels of free cytoplasmic Ca⁺⁺. (iii) The saturating depolarization is only about 30% as large as the maximal receptor potentials of contralateral, dark controls, and by that measure the log V-log I curve is shifted downward by 0.54 log units. The gain (change in conductance per absorbed photon) therefore appears to have been diminished.     

The 9-methyl group of retinal is essential for rapid Meta II decay and phototransduction quenching in red cones

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.     

Photoconvertible pigment states and excitation in Calliphora; the induction and properties of the prolonged depolarising afterpotential

1. The proposed models of two independent groups, which relate the different states of the visual pigment to the excitation of the membrane in invertebrate photoreceptors (with particular reference to the prolonged depolarising afterpotential, the PDA) are compared and evaluated. 2. The validity of the late receptor potential (the "normal" receptor response) as an index of photoreceptor sensitivity, i.e., an index of the number of rhodopsin to metarhodopsin transitions, is verified by concurrent spectrophotometry. 3. Electrophysiological observations alone allow the calculation of 1.3 x 10(8) photopigment molecules in the rhabdom of an R1-6 photoreceptor of a vitamin A-bred Calliphora. 4. The PDA is shown to be quantifiable in terms of the number of rhodopsin to metarhodopsin conversions by the absorption of single light quanta. 5. The comparison of discrete membrane fluctuations (quantum bumps) during the PDA and during exposure to sustained light stimuli that mimic the PDA suggest that, the PDA, similar to the late receptor potential, may be due to the summation of quantum bumps.     

Properties of the pH-sensitive site that controls the lambda max of Limulus metarhodopsin

A pH-sensitive site controls the lambda max of Limulus metarhodopsin. The properties of this site were examined using intracellular recordings of the early receptor potential (ERP) as a pigment assay. ERPs recorded over a range of extracellular pHs indicate that the apparent pK of the site is in the range of 8.3-8.6. Several lines of evidence indicate that the site responds directly to changes in extracellular pH (pHo) rather than to changes in intracellular pH(pHi) that follow as a secondary result of changing pHo : (a) the effect of changing pHo was rapid (less than 60 s); (b) when pHo was raised, the simultaneous rise in pHi, as measured with phenol red, was relatively small; (c) raising pHi by intracellular injection of pH 10 glycine buffer did not affect the site; and (d) the effect of changing pH0 could not be blocked by increasing the intracellular pH buffering capacity. It is concluded that the pH-sensitive site on metarhodopsin is on the extracellular surface of the plasma membrane.     

Removal of the 9-methyl group of retinal inhibits signal transduction in the visual process. A Fourier transform infrared and biochemical investigation

The photoreaction of opsin regenerated with 9-demethylretinal has been investigated by UV-vis spectroscopy, flash photolysis experiments, and Fourier transform infrared difference spectroscopy. In addition, the capability of the illuminated pigment to activate the retinal G-protein has been tested. The photoproduct, which can be stabilized at 77 K, resembles more the lumirhodopsin species, and only minor further changes occur upon warming the sample to 170 K (stabilizing lumirhodopsin). UV-vis spectroscopy reveals no further changes at 240 K (stabilizing metarhodopsin I), but infrared difference spectroscopy shows that the protein as well as the chromophore undergoes further molecular changes which are, however, different from those observed for unmodified metarhodopsin I. UV-vis spectroscopy, flash photolysis experiments, and infrared difference spectroscopy demonstrate that an intermediate different from metarhodopsin II is produced at room temperature, of which the Schiff base is still protonated. The illuminated pigment was able to activate G-protein, as assayed by monitoring the exchange of GDP for GTP gamma S in purified G-protein, only to a very limited extent (approximately 8% as compared to rhodopsin). The results are interpreted in terms of a specific steric interaction of the 9-methyl group of the retinal in rhodopsin with the protein, which is required to initiate the molecular changes necessary for G-protein activation. The residual activation suggests a conformer of the photolyzed pigment which mimics metarhodopsin II to a very limited extent.