Correspondence to: Hugh R. Matthews, Physiological Laboratory, Downing Street, Cambridge CB2 3EG, United Kingdom. Fax:44-223-333840 E-mail:hrm1{at}cam.ac.uk.
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Abstract |
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During adaptation Ca2+ acts on a step early in phototransduction, which is normally available for only a brief period after excitation. To investigate the identity of this step, we studied the effect of the light-induced decline in intracellular Ca2+ concentration on the response to a bright flash in normal rods, and in rods bleached and regenerated with 11-cis 9-demethylretinal, which forms a photopigment with a prolonged photoactivated lifetime. Changes in cytoplasmic Ca2+ were opposed by rapid superfusion of the outer segment with a 0Na+/0Ca2+ solution designed to minimize Ca2+ fluxes across the surface membrane. After regeneration of a bleached rod with 9-demethlyretinal, the response in Ringer's to a 440-nm bright flash was prolonged in comparison with the unbleached control, and the response remained in saturation for 1015s. If the dynamic fall in Ca2+i induced by the flash was delayed by stepping the outer segment to 0Na+/0Ca2+ solution just before the flash and returning it to Ringer's shortly before recovery, then the response saturation was prolonged further, increasing linearly by 0.41 ± 0.01 of the time spent in this solution. In contrast, even long exposures to 0Na+/0Ca2+ solution of rods containing native photopigment evoked only a modest response prolongation on the return to Ringer's. Furthermore, if the rod was preexposed to steady subsaturating light, thereby reducing the cytoplasmic calcium concentration, then the prolongation of the bright flash response evoked by 0Na+/0Ca2+ solution was reduced in a graded manner with increasing background intensity. These results indicate that altering the chromophore of rhodopsin prolongs the time course of the Ca2+-dependent step early in the transduction cascade so that it dominates response recovery, and suggest that it is associated with photopigment quenching by phosphorylation.
Key Words: retinal rod, calcium, photoreceptor, rhodopsin, adaptation
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INTRODUCTION |
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It is well established that the cytoplasmic calcium concentration (Ca2+i) plays an important role in the modulation of transduction in vertebrate photoreceptors during light and dark adaptation (for reviews see
Photoreceptor Ca2+i is believed to be governed in darkness by the balance between Ca2+ influx through the outer segment conductance (0.5 s (
The molecular basis for the actions of Ca2+ early in the transduction cascade is still the subject of some debate (for review see
To investigate the identity of this Ca2+-sensitive step, we studied the effect of delaying the light-induced decline in Ca2+i after a bright flash in rods that had been bleached and regenerated with 11-cis 9-demethylretinal. This retinal analogue has been shown to form a photopigment that exhibits a prolonged photoactivated lifetime (
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MATERIALS AND METHODS |
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Preparation
Details of the preparation, recording techniques, light stimuli, and solution changes have been described previously (80 ms, measured as the time for the junction current to rise from 10 to 90% of its final level. All experiments were performed at room temperature (
20°C).
External Solutions
Ringer's solution contained the following: 111 mM NaCl, 2.5 mM KCl, 1.0 mM CaCl2, 1.6 mM MgCl2 and 3.0 mM HEPES, adjusted to pH 7.7 with NaOH, and also included 10 µM EDTA to chelate impurity heavy metal ions. The Ringer's solution perfusing the recording chamber also included 10 mM glucose. 0Na+/0Ca2+ solution was modified from this composition by the equimolar substitution of choline chloride (Sigma-Aldrich) for NaCl, the inclusion of 2 mM EGTA and omission of CaCl2 and MgCl2 to reduce the divalent cation concentration to extremely low levels, the titration of the HEPES buffer with tetramethylammonium hydroxide instead of NaOH, and the omission of EDTA. This solution served to oppose light-induced changes in Ca2+i by simultaneously minimizing Ca2+ influx and efflux (
Light Stimuli and Electrical Recording
Light stimuli were delivered from a dual-beam optical stimulator controlled by electromagnetic shutters. The wavelength of stimulation was selected using interference filters (bandwidth 10 nm for 440-nm and 40 nm for 650-nm center wavelength); flash stimuli were of 20 ms duration and unpolarized. Stimulus intensities were adjusted with neutral density filters and measured with a calibrated silicon photodiode (Graseby Optronics). Dark-adapted rods were stimulated with 500 nm light, to excite the native photopigment, which absorbs maximally at 516 nm (
The photopigment formed upon regeneration with 9-demethylretinal absorbs maximally at 465 nm (-band of photoreceptor spectral sensitivity curves (
185 times as effectively as by the analogue pigment. However, this value needs to be corrected to take account of the estimated proportion of native pigment remaining after bleaching and the relative efficacy of the two photopigments in exciting transduction. The analogue pigment appears to evoke a quantal response some 30 times smaller than does the native pigment (
28 times more effective at stimulating transduction via the native than via the analogue pigment. In contrast, 440-nm light would be predicted to be absorbed a little over twice as effectively by the analogue than by the native pigment. So if even as much as 0.5% of the native pigment remained after bleaching, then 440-nm light would be expected to be at least 15 times as effective at stimulating transduction via the analogue as via the native pigment.
The suction pipette current signal was filtered over the bandwidth DC-20 Hz (Bessel filter), and digitized continuously for subsequent analysis at a sampling rate of 100 Hz using an IBM-compatible microcomputer equipped with an intelligent interface card (Cambridge Research Systems).
Regeneration of the Photopigment after Bleaching
After bleaching, the photopigment was regenerated by the addition to the recording chamber (volume 200 µl) of 200 µl of a suspension of phospholipid vesicles containing either 11-cis retinal or 11-cis 9-demethylretinal. Experiments with heterologously expressed wild-type opsin (
The 9-demethylretinal was synthesized as described previously (
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RESULTS |
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The procedure used to form the 9-demethylretinal photopigment within an isolated rod is illustrated in Fig 1 A. First, the outer segment was exposed for 5 min to intense light calculated to bleach in excess of 99% of the photopigment. This resulted in the rapid and complete suppression of the circulating current recorded by the suction pipette. Next, a suspension of phospholipid vesicles containing 9-demethylretinal was introduced into the recording chamber, leading to the gradual recovery of the circulating current over the next 40 min as the analogue photopigment was formed (
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After regeneration with 9-demethylretinal, the light response was no longer spectrally univariant (
In cells regenerated with 9-demethylretinal, the final recovery of responses to bright flashes (as used in later figures) at 440 nm took place extremely slowly, requiring 5 min or more for the complete return to baseline and necessitating a similarly long interval between trials. Due to the considerably greater efficacy of this short wavelength in stimulating transduction via the analogue than via the native pigment and the much more rapid kinetics of the response evoked by the native pigment, the recovery from saturation of the response to a 440-nm bright flash seems likely to have been dominated by the quenching of the analogue photopigment. However, the responses to very bright flashes at 650 nm also often exhibited a slow final component of recovery. This observation suggests that, even at this unfavorably long wavelength, enough of the analogue photopigment could be activated by a flash of sufficiently high intensity to contribute to response recovery at later times when the native pigment would have already been quenched.
Fig 2 examines the effect of stepping the outer segment of a cell which had been bleached and then regenerated with 9-demethylretinal into 0Na+/0Ca2+ solution 1 s before a bright flash, and remaining in this solution for a progressively increasing period thereafter. The flash was of wavelength 440 nm, to evoke a response whose recovery from saturation will have been dominated by the excitation of the analogue photopigment. Since most permeant cations have been replaced by the impermeant cation choline, 0Na+/0Ca2+ solution supports an inverted dark current that is carried predominantly by K+ (
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This progressive increase in response duration when the dynamic fall in Ca2+i was delayed after regeneration with 9-demethylretinal can be contrasted with the results shown in Fig 3, which were obtained from a bleached rod regenerated with 11-cis-retinal, using bright flashes of wavelength 500 nm to best stimulate the regenerated rhodopsin. In this case, the recovery of the flash response was little affected by the solution change unless the time spent in 0Na+/0Ca2+ solution exceeded the normal duration of the flash response in Ringer's (Fig 3, traces labeled R). Similarly, modest effects of exposure to 0Na+/0Ca2+ solution were obtained from a second bleached rod regenerated with 11-cis-retinal and also from five unbleached dark-adapted rods. These results are directly comparable to those obtained previously from dark-adapted rods using a rather lower flash intensity (
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These results are quantified in Fig 4 by measuring the time taken after the flash for the response to recover 25% of the original dark current in Ringer's. The prolongation of the response after exposure to 0Na+/0Ca2+ relative to the response to the same flash in Ringer's is plotted in Fig 4 A as a function of the time spent in 0Na+/0Ca2+ solution after the flash. In each case, these data have been normalized according to the time for 25% response recovery for the same cell in Ringer's. In unbleached control cells (open symbols) and in bleached cells regenerated with 11-cis-retinal (half-closed symbols), exposures to 0Na+/0Ca2+ solution, which ended before the response in Ringer's, would normally have recovered had only a modest effect on the duration of the response. Even when the exposure extended beyond the normal time for 25% recovery in Ringer's, the response was prolonged by little more than the additional time spent in 0Na+/0Ca2+ solution after the flash. These results are comparable to the modest slowing of the response that has been observed previously in dark-adapted rods upon delaying the dynamic fall in Ca2+i after a dimmer but still saturating flash (see Fig 4 B of
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Fig 4 B shows mean data from the same cells plotted in absolute coordinates. In unbleached cells (open symbols) exposure to 0Na+/0Ca2+ solution resulted in only a modest retardation of response recovery. However, in bleached cells regenerated with 9-demethylretinal (closed symbols), the mean duration of the response increased linearly with the time spent in 0Na+/0Ca2+ solution after the flash, and could be well fitted by a regression line of slope 0.41 ± 0.01 over the full range of exposure durations. This observation indicates that, after regeneration with 9-demethylretinal, recovery remains Ca2+-sensitive for the entire duration of the response, suggesting that the Ca2+-sensitive step early in transduction has been prolonged to such an extent that it now dominates response recovery. If this were the case, then it would imply that response recovery after excitation of the analogue photopigment should vary in a graded manner with Ca2+i. This possibility was addressed by using steady light to reduce Ca2+i, and then examining the degree of response prolongation evoked by 0Na+/0Ca2+ solution in a bleached cell regenerated with 9-demethylretinal.
Results from such an experiment are illustrated in Fig 5. First, the cell was exposed to subsaturating steady illumination in Ringer's for 14 s to reduce Ca2+i from its value in darkness to a light-adapted level that will have varied in a graded manner with the proportion of current suppressed (12 s, the outer segment was returned to Ringer's before the onset of response recovery. This procedure was intended to hold Ca2+i at a reduced level for a fixed period during the period of response saturation. These traces are compared with responses to the same illumination sequence for which the outer segment remained in Ringer's throughout. In the absence of prior background illumination (Fig 5 A), this procedure resulted in a substantial prolongation of the bright flash response in comparison with the response in Ringer's (Fig 5, trace labeled R) as in previous figures. However, as the background intensity was increased (Fig 5B and Fig C), the response was prolonged by a progressively shorter period until, for near-saturating light (Fig 5 D), the degree of response prolongation was very small.
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Results from a total of six such experiments are collected in Fig 6, in which the degree of response prolongation is plotted as a function of the circulating current remaining for each background intensity at the time of the step to 0Na+/0Ca2+ solution. Since Ca2+i has been shown to vary progressively with the circulating current (
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However, the data also admit to the alternative interpretation that, instead of being governed by the level of Ca2+i during the initial part of the response while the outer segment remained in 0Na+/0Ca2+ solution, the absolute prolongation of the response to this flash of fixed intensity might depend instead upon its duration, which also was reduced progressively during adaptation as the background intensity increased. This possibility was investigated by examining (Fig 7) the response prolongation evoked by a constant duration exposure to 0Na+/0Ca2+ solution of a bleached cell regenerated with 9-demethylretinal for flashes of differing intensity. The almost 20-fold increase in flash intensity in Fig 7 (AC) led to a near doubling in the duration of the flash response, together with progressive changes in the waveform of response recovery. Nevertheless, comparison of these responses reveals that exposure to 0Na+/0Ca2+ solution evoked a near-constant prolongation for the recovery to a criterion level relative to that in Ringer's (Fig 7, traces labeled R) irrespective of the absolute response duration or flash intensity.
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Results from three such experiments are collected in Fig 8, in which is plotted the time for 25% recovery of the original dark current after exposure to in 0Na+/0Ca2+ solution against the time for 25% recovery for the same flash in Ringer's. These points fall on a straight line that is nearly parallel to the line of unit slope. This observation indicates that exposure to 0Na+/0Ca2+ solution for a fixed period evokes a near-constant delay in response recovery, irrespective of the intensity of the flash. This observation supports the notion that the graded variation in response prolongation by 0Na+/0Ca2+ solution that was observed when Ca2+i was manipulated with steady light resulted from a graded variation in the time constant governing response recovery after stimulation of the analogue photopigment. The slight deviation from unit slope may reflect the progressive departure from translational invariance of the recovery waveforms in Ringer's as the flash intensity was increased (
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DISCUSSION |
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Prolongation of Ca2+ Sensitivity by 9-Demethylretinal
In rods bleached and regenerated with 9-demethylretinal, the recovery of the response to a bright flash was found to depend strongly on the timing of the associated changes in Ca2+i. When the fall in Ca2+i, which accompanies the response to a bright flash, was delayed by progressively longer exposures to 0Na+/0Ca2+ solution, the response was prolonged in a graded manner throughout its entire duration (Fig 2 and Fig 4). This contrasts with the much more modest effect of delaying the light-induced fall in Ca2+i under control conditions (
These results indicate that as well as retarding the recovery of the flash response (0.5 s (
Quantitative Description of the Actions of Ca2+ Early in Transduction
The extent to which this now dominant time constant is modulated by Ca2+ in rods regenerated with 9-demethylretinal can be estimated from the relationship between the time spent in 0Na+/0Ca2+ solution and the prolongation of the bright flash response (Fig 4 B). A conceptual model of this process is illustrated in Fig 9. Suppose that the time constant that governs the exponential decay of some Ca2+-sensitive intermediate early in the transduction cascade has been prolonged by 9-demethylretinal to such a degree that it now dominates the kinetics of response recovery. In Ringer's solution, Ca2+i would fall rapidly after the flash, so that this intermediate would experience a greatly lowered Ca2+ concentration for virtually the entire duration of the response, and, therefore, would decay rapidly (Fig 9, heavy trace, time constant f). If instead the outer segment were exposed to 0Na+/0Ca2+ solution for a period (T) after the flash, then Ca2+i would remain near to its initial value in darkness and this intermediate would decay more slowly (Fig 9, light trace, time constant
s). However, once the outer segment was returned to Ringer's solution Ca2+i would fall rapidly and the decay would accelerate to match that in the absence of the solution change. In reality, Ca2+i would not fall instantaneously, but if it declined with similar kinetics in Ringer's irrespective of whether the flash was delivered in Ringer's or in 0Na+/0Ca2+ solution, then the time course of intermediate decay would be likely to be affected in an equivalent manner in both cases.
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On the basis of this simple model, the activity of the Ca2+-sensitive intermediate would be predicted to recover to the level at the time of the solution change according to Equation 1 when:
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(1) |
where T is the time spent in 0Na+/0Ca2+ solution, and T is the additional delay before the intermediate decays to the level that it would have reached at time T in Ringer's solution. Solving for
T:
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(2) |
Equation 2 predicts that the additional delay before recovery to a criterion level should increase in direct proportion to the time spent in 0Na+/0Ca2+ solution after the flash, as observed in Fig 4. The precise criterion level selected is unimportant, due to the translational invariance of both the exponential curves of the model and the recovery phase of the bright flash responses in Ringer's and 0Na+/0Ca2+ solution. The slope of 0.41 for the regression line fitted to the data of Fig 4 B yields a value for the ratio of the fully speeded (f) to the resting (
s) time constants of 0.59, implying that the dynamic reduction of Ca2+i from its dark-adapted value during the flash response can speed this Ca2+-sensitive step by a factor of 1.7 for this analogue photopigment.
Site of Action of Ca2+ Early in Transduction
When a bleached rod is exposed to 9-demethylretinal, an analogue photopigment is formed with modified spectral sensitivity and a prolonged photoactivated lifetime (
How might the extended functional lifetime of the photopigment formed with 9-demethylretinal arise? One possible solution to this question is provided by the recent demonstration that when 9-demethylrhodopsin is excited by light, the equilibrium between the meta-I and meta-II forms of the analogue photopigment overwhelmingly favors the meta-I form (
A second possibility is that the prolonged functional lifetime of the analogue photopigment might reflect differences in the quenching of its active form when compared with native rhodopsin. For example, the photopigment formed with 9-demethylretinal exhibits reduced light-dependent phosphorylation when compared with the native photopigment (
Irrespective of whether one or both of these possibilities applies, the prolongation of the period of Ca2+ sensitivity after a flash and the modulation of the extent of this prolongation by Ca2+i indicate that the functional lifetime of the form of the photoactivated analogue pigment directly preceding the Ca2+-sensitive quenching step is prolonged. This argues against a crucial role for impaired arrestin binding to phosphorylated 9-demethylrhodopsin, since this process is not believed to be Ca2+ dependent. In contrast, the phosphorylation of photoisomerized rhodopsin is believed to depend on Ca2+i through the actions of recoverin on rhodopsin kinase (
It has been suggested that in addition to modulating the lifetime of an early photoproduct, the light-induced fall in Ca2+i might also lead to a change in the gain with which photoisomerized rhodopsin activates the transduction cascade (
Functional Implications
The identification of the Ca2+-sensitive step early in phototransduction with photopigment quenching has a number of functional implications. First, it resolves the ambiguity in assigning the fast and slow time constants that shape the light response to specific processes (
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Acknowledgements |
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We thank Dr. John Oatis for help with the retinoid synthesis and Dr. G.L. Fain for helpful discussions.
This study was supported by the Wellcome Trust, the National Eye Institute of the National Institutes of Health (EY01157 [to M.C. Cornwall] and EY04939 [to R.K. Crouch]), the Foundation Fighting Blindness, and an unrestricted grant from Research to Prevent Blindness, Inc., to the Department of Ophthalmology at Medical University of South Carolina.
Submitted: 18 May 2001
Revised: 10 August 2001
Accepted: 21 August 2001
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