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Physiological Laboratory, University of Cambridge, Downing Street, Cambridge, England CB2 3EG; and Department of Physiology, Boston University Medical School, Boston, Massachusetts 02118
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ABSTRACT |
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Key Words: photoreceptor • retinal rod • bleaching adaptation • calcium
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introduction |
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TopABSTRACTintroductionmethodsresultsdiscussionreferences |
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; Cornwall et al., 1990). The persistent suppression of the circulating current after bleaching results from a continuing excitation of phototransduction (Cornwall and Fain, 1994; Cornwall et al., 1995) via excitation of the transduction cascade (Matthews et al., 1996a) by a long-lived photoproduct of rhodopsin bleaching that may be free opsin (Jin et al., 1993).
Bleaching adaptation shows considerable similarity with the adaptational changes induced by steady light (Fain and Cornwall, 1993; Leibrock et al., 1994; Jones et al., 1996). Such light adaptation is believed to be largely, if not exclusively, mediated by a light-induced fall in Ca2+i, which results from the graded suppression of the circulating current by the background, since if these changes in Ca2+i are prevented by interfering with the normal mechanism of Ca2+ homeostasis then adaptation is abolished (Matthews et al., 1988; Nakatani and Yau, 1988; Fain et al., 1989). Similar manipulations of Ca2+ homeostasis have also been shown to prevent these bleach-induced adaptational changes in cone photoreceptors (Matthews et al., 1996c), suggesting that Ca2+i may also play a similar role in bleaching adaptation.
The fall in Ca2+i that immediately follows the suppression of the circulating current by light has been measured in several recent studies, which demonstrates that Ca2+i varies in a graded manner during steady illumination (Gray-Keller and Detwiler, 1994, 1996; McCarthy et al., 1994; Younger et al., 1996). However, considerably less is known about the changes in Ca2+ that may take place during the photopigment cycle. We have therefore applied a laser spot confocal technique, originally developed for measuring the release of Ca2+ from sarcoplasmic reticulum (Escobar et al., 1994), to the measurement of light-induced changes in Ca2+i from salamander rod photoreceptors. In common with previous studies using fluorescent probes to measure changes in Ca2+i from single rods, the fluorescence excitation beam resulted in considerable stimulation of the photoreceptor. Making a virtue of this necessity, we have used the photopigment bleaching that takes place during the Ca2+ measurement itself to follow the changes in Ca2+i that accompany photopigment bleaching and regeneration in isolated rod photoreceptors. Preliminary results from this study have been reported to the Physiological Society (Matthews et al., 1996b) and to the Association for Research in Vision and Ophthalmology (Sampath et al., 1997).
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Ca2+ Measurement
Simultaneous measurements of photocurrent and Ca2+ indicator fluorescence were made with a spot confocal technique (Escobar et al., 1994) illustrated schematically in Fig. 1. The beam from an argon ion laser (Model 98; Lexel Laser Inc., Freemont, CA), tuned to 514 nm, illuminated the 50-µm pinhole of a spatial filter assembly (Newport Corp., Irvine, CA). The beam was recollimated by a 4x microscope objective (Nikon Inc., Melville, NY), and entered the inverted microscope (Zeiss IM; Carl Zeiss, Inc., Oberkochen, Germany) through the epi-fluorescence port via a dichroic mirror (525DRLP; Omega Optical, Brattleboro, VT). A 40x objective lens (1.3 NA oil immersion; Nikon Inc.) formed a reduced image of the pinhole to illuminate a spot 
8 µm in diameter on the outer segment of a rod photoreceptor, whose inner segment was drawn into a suction pipette. The fluo-3 fluorescence evoked by laser illumination passed through a 530-nm emission filter (530EFLP; Omega Optical) and was imaged onto a small-diameter PIN photodiode (model HR008; United Detector Technologies, Hawthorne, CA) mounted on the headstage of an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). The analog signal from the diode was low pass filtered at 250 Hz with an active 8-pole Bessel filter (Frequency Devices, Haverhill, MA) and digitized at 1,000 Hz (PClamp 6; Axon Instruments).
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In Situ Calibration
To quantify how photoreceptor Ca2+ concentration changes during exposure to light, both the Kd of fluo-3 and the maximum and minimum fluorescence that can be elicited from the rod outer segment in high and low Ca2+ (Fmax and Fmin) are required (Minta et al., 1989). The Kd of the Ca2+ indicator was determined with a haemocytometer (100 µm path length) containing 100 µM fluo-3 free acid in pseudo-intracellular solutions of buffered Ca2+ (World Precision Instruments, Sarasota, FL), whose precise free Ca2+ concentrations were verified using a Ca2+ electrode (Calcium Ionophore I–cocktail A; Fluka, Ronkonkoma, NY). The sigmoidal variation of the fluorescence signal (F) with free Ca2+ concentration (Fig. 1, inset) could be fitted by the Michaelis-Menten equation for Ca2+ binding:
 | (1) |
which yielded a value for Kd of 400 nM.
Fmax and Fmin were determined in situ using the four-laser pulse protocol described above while exposing the photoreceptor outer segment to solutions of low and high Ca2+ concentration containing 25 µM ionomycin or 40 mg/ml saponin. The low Ca2+i solution that was used to determine Fmin consisted of 111 mM NaCl, 2.5 mM KCl, 2.05 mM MgCl2, 3 mM HEPES, and 2 mM EGTA. The high Ca2+ solution that was used to determine Fmax contained 76.6 mM CaCl2, 2.5 mM KCl, and 3 mM HEPES. In both cases, the pH was titrated to 7.7 with NaOH. Values of Fmin and Fmax measured in this way were used in Eq. 1 to determine the free Ca2+ concentration in the same dark-adapted rod before and after exposure to saturating laser illumination. The value of Fmin determined in situ was a greater fraction of Fmax than was obtained from fluo-3 in the cuvette (see Fig. 1, inset); this discrepancy may reflect nonspecific binding of the dye within the rod outer segment (see DISCUSSION).
Fluo-3-free Acid
To ensure that the fluorescence signal measured with fluo-3 AM was not contaminated by compartmentalization of the dye within the outer segment, Ca2+ measurements were also made with the cell-impermeant fluo-3-free acid incorporated into the rod cytoplasm by whole-cell patch-clamp recording. Patch pipettes with tip resistances between 5 and 12 M
were filled with a pseudo-intracellular solution (92 mM KAspartate, 7 mM NaCl, 5 mM MgCl2, 10 mM HEPES, 1 mM Na2ATP, 1 mM Na4GTP, pH 7.0) that included 100 µM fluo-3 (pentapotassium salt). Gigaohm seals were formed on the rod inner segment, and the whole-cell recording configuration was established by either gentle suction or hyperpolarizing voltage clamp pulses so as to allow access of the pipette solution to the cytoplasm by diffusion. The membrane potential in the whole-cell configuration was held at –40 mV (after correction for a 10-mV liquid junction potential).
Electrical Recording and Light Stimuli
Light-induced changes in the circulating current were recorded from the suction or patch pipette with an EPC-7 patch clamp amplifier (List Electronics, Darmstadt, Germany). This signal was low pass filtered at 20 Hz using an active 8-pole Bessel filter (Frequency Devices) and digitized either at 100 or 1,000 Hz (for simultaneous photocurrent and calcium indicator fluorescence measurements).
Light stimuli were delivered from a DC-driven tungsten halogen lamp through a 570-nm interference filter, and unpolarized; flashes of 20-ms duration were delivered through electromagnetic shutters (Vincent Associates, Rochester, NY). Light intensities were adjusted with neutral density filters (Fish-Schurman Corp., New Rochelle, NY) and calibrated with a silicon photodiode (Graseby Optronics, Orlando, FL). These absolute photon fluxes can be converted to rates of rhodopsin isomerization using a collecting area of 7 µm2 at 570 nm, obtained by multiplying the value of 20 µm2 (Lamb et al., 1986) at the 516-nm wavelength of peak sensitivity (Harosi, 1975) by the ratio of the spectral sensitivities of the rod photopigment at these two wavelengths (Cornwall et al., 1984).
The percentage of photopigment bleached by the 514 nm light from the argon ion laser was estimated from the photosensitivity at 520 nm for vitamin A2–based pigments in free solution (Dartnall, 1972), corrected for the difference in dichroism in free solution and in disk membranes (6.2 x 10–9 µm2) (Jones et al., 1993). Both the continuous and the short pulse laser exposures that were used to measure the light-induced decline in Ca2+i can be calculated from the photosensitivity to bleach >99% of the photopigment within the area of the laser spot, although this may have been reduced somewhat in practice by photoregeneration from bleaching intermediates, especially for the short pulses (Williams, 1964; Pugh, 1975). Less extensive bleaching is also likely to have taken place in the remainder of the outer segment due to scattered laser light.
Pigment Regeneration
Pigment regeneration was accomplished by bath application of phospholipid vesicles containing the chromophore 11-cis-retinal. A 25-mg aliquot of phosphatidyl choline solution (Type V; Sigma Chemical Co.) was dried in a stream of 100% N2. To this was added 10 ml of Ringer solution without glucose or BSA, and the mixture was sonicated for 10 min (60 W, 50% duty cycle) with a probe sonicator (Vibracell; Sonics Materials Inc., Danbury, CT). The resulting vesicle suspension was then added to a small vessel containing 11-cis-retinal dried on its wall from an ethanolic solution. Loading of the vesicles with the retinoid was facilitated by further brief sonication in darkness. The concentration of 11-cis-retinal present in the vesicles was measured spectrophotometrically (UV-2101PC; Shimadzu Scientific Instruments, Columbia, MD) using the molar extinction coefficient in ethanol of 25,000 M–1 cm–1 at 380 nm (Hubbard et al., 1971), and the vesicle suspension was diluted with Ringer solution to a final concentration of 300 µM. The photopigment of bleached rods was regenerated by adding a sufficient quantity of the vesicle suspension to the recording chamber to achieve a final 11-cis-retinal concentration of 100 µM.
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; McNaughton et al., 1986; Ratto et al., 1988).
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In these calibration measurements, the ratio Fmax/ Fmin was consistently less than that obtained in the cuvette (see Fig. 1, inset). This observation suggested either that a residual "pedestal" of Ca2+-insensitive fluorescence contributed to Fmin, or alternatively that Fmax was consistently underestimated in these experiments. To address the second possibility that superfusion with high Ca2+ solution in the presence of ionomycin might not have raised Ca2+i sufficiently to completely saturate fluo-3 fluorescence, similar measurements were carried out in which saponin was used instead of ionomycin to disrupt the outer segment membrane more extensively. While there was little difference between the results obtained with saponin and ionomycin in zero-Ca2+ solution, superfusion with high Ca2+ solution led to a similar initial rise that was followed by a further delayed increase in fluorescence in the presence of saponin. This second component of the fluorescence increase seems most likely to have represented the release of fluo-3 compartmentalized or bound within the outer or inner segments (Schnetkamp et al., 1991b), although the possibility that ionomycin and isotonic Ca2+ may have failed to saturate dye fluorescence cannot be completely excluded. We therefore interpret the reduced ratio Fmax/Fmin as representing a residual Ca2+- and light-insensitive pedestal of fluorescence, which may represent fluo-3 bound or compartmentalized within the rod outer segment (McCarthy et al., 1994).
To address the possibility that the light-induced decay of the fluo-3 fluorescence signal might have originated from membrane-permeant fluo-3 AM, which had entered a subcellular compartment other than the cytoplasm, experiments were carried out in which the membrane-impermeant fluo-3-free acid was incorporated into the cytoplasm from a patch pipette. Fig. 5 compares the fluorescence responses from rods loaded by incubation with fluo-3 AM (Fig. 5 A), and by 4 min of whole cell recording with a patch pipette containing an artificial intracellular solution that included 100 µM fluo-3 pentapotassium salt (Fig. 5 B). It can be seen that the fluorescence signal declined in a similar manner during the first laser exposure (Fig. 5, trace 1) and remained depressed for the second (Fig. 5, trace 2), irrespective of the means by which fluo-3 had been incorporated. In Fig. 5 B, a slight decay in fluorescence occurred on the second laser exposure (trace 2); the origin of this residual relaxation is not clear. Nevertheless, after complete suppression of the circulating current, a comparable level of residual fluorescence remained in both cases, despite the fact that the membrane-impermeant form of fluo-3 would be expected to be less readily compartmentalized. Furthermore, qualitatively similar results were obtained in experiments in which incubation with fluo-3 AM was carried out at 4°C instead of room temperature, a procedure that is believed to decrease dye compartmentalization. These observations suggest that the residual pedestal of fluorescence may have originated instead from nonspecific binding of the dye within the outer segment cytoplasm (McCarthy et al., 1994).
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Changes in Ca2+i Induced by Bleaching
The principal objective of this study was to investigate how Ca2+i changes in the rod outer segment after photopigment bleaching. This was achieved by making repeated measurements of fluo-3 fluorescence from a single salamander rod when dark adapted, after exposure to laser light sufficiently intense to bleach a substantial fraction of the photopigment, and after regeneration with 11-cis-retinal, as illustrated in Figs. 6 and 7. First, the sensitivity of the dark-adapted rod was measured with dim flashes. Then laser pulses were delivered to investigate the decline of Ca2+i from its dark-adapted level (Fig. 6 A). The corresponding fluorescence intensity evoked by the first laser pulse has been plotted in Fig. 7 C, together with the level to which it declined after the bleach-induced suppression of the dark current. These values reflect relative Ca2+i levels before and immediately after the bleaching laser exposure. These two sequences of laser illumination, each consisting of four 20-ms laser pulses, are calculated to have bleached >99% of the photopigment within the area of the laser spot (see METHODS), while scattered laser light is likely to have caused significant bleaching within the remainder of the outer segment.
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As noted above, a significant light- and Ca2+-insensitive pedestal of fluorescence remained after complete suppression of the circulating current. In some cells, this pedestal increased somewhat during the prolonged time course of the experiment. This may have reflected entry into the outer segment of dye previously compartmentalized in the inner segment, or slight movement of the outer segment relative to the laser spot. To attempt to compensate for this variability, the data were also analyzed by dividing the peak fluo-3 fluorescence for each cell by the corresponding value for this pedestal. When analyzed in this way, the normalized peak fluorescence fell significantly from 3.7 ± 0.4 times the pedestal value at the start of the experiment to 2.4 ± 0.3 after bleaching by the laser spot; a decrease of 35%. However, after regeneration of the photopigment with 11-cis-retinal, the normalized peak fluorescence rose again to 3.4 ± 0.4, a value not significantly different from the original level in darkness. These measurements indicate that when the dark current and sensitivity were depressed after bleaching, the fluo-3 fluorescence signal was depressed also, whereas regeneration of the photopigment with 11-cis-retinal restored all of these parameters to near their original dark-adapted values.
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; Minta et al., 1989). Furthermore, the accuracy of the measurement is not compromised at values of Ca2+i much lower than the dissociation constant, as would be the case for a ratiometric Ca2+ indicator. Second, the confocally imaged laser spot can be used to excite and then collect dye fluorescence from a well-defined region of the outer segment (Escobar et al., 1994). This is of particular importance in the intact rod since the ellipsoid region of the inner segment loads heavily with fluo-3 AM and might otherwise contribute to the fluorescence signal. Finally, this method can also be used for cells with much smaller outer segments than the salamander rods employed in this study, such as amphibian cones (Matthews et al., 1996b) and mammalian rods. A potential drawback of this single-wavelength measurement is that if significant fluo-3 bleaching were to take place during the laser exposure, the corresponding change in fluorescence would be indistinguishable from that caused by a real decrease in Ca2+i. However, several pieces of evidence indicate that significant dye bleaching is unlikely to have taken place during these measurements. First, if the total laser exposure is substantially reduced, either by attenuating the laser beam intensity or by presenting repeated brief laser pulses instead of a continuous exposure, then the time course of the subsequent decline in fluorescence is little affected. These approaches will each have reduced the laser exposure by at least a further order of magnitude. Second, if after the initial laser exposure the laser spot is displaced to a region of the outer segment that has not previously been excited directly, then fluo-3 fluorescence is depressed there also, even though the dye in this region has not previously been exposed to the laser spot. Third, repeated laser exposures show that fluo-3 fluorescence remains depressed after the first exposure of a dark-adapted rod to the laser and does not show a further relaxation within the time course of our measurements, consistent with the notion that Ca2+i has already reached its minimum level once the circulating current has been completely suppressed. Furthermore, the subsequent recovery of the fluorescence signal appears to take place in parallel with the gradual recovery of the dark current after photopigment bleaching. Finally, application of exogenous 11-cis-retinal restores the fluorescence signal along with circulating current and sensitivity. Taken together, these observations indicate that the decline in fluo-3 fluorescence during saturating illumination of a dark-adapted rod is a real physiological phenomenon and not an artifact of fluo-3 bleaching.
The excitation and emission wavelengths for fluo-3 fall relatively close to the peak of the absorbance spectrum for rhodopsin. Nonetheless, bleach-induced changes in self-screening by rhodopsin or its photoproducts seems unlikely to have affected the intensity of fluo-3 fluorescence significantly for two reasons. First, the absolute magnitude of such self-screening is relatively small, corresponding to little over 10% for randomly polarized transverse illumination of a dark-adapted salamander rod (Harosi, 1975). Second, the exciting beam was sufficiently intense that the vast majority of the photopigment within the area of the laser spot will have been bleached within a few milliseconds of the initial laser exposure. Subsequent fluorescence measurements taken a few seconds later once the circulating current was fully suppressed were made at a time when meta III, the only photoproduct that absorbs at wavelengths close to those of fluorescence excitation or emission, will not yet have appeared (Baumann, 1972; Donner and Hemilä, 1975). In contrast, tens of minutes later when Ca2+i was measured again once the responses of the bleached rod had stabilized, most of the meta III produced by the initial laser exposure will subsequently have decayed, and therefore will not have contributed to self-screening.
Light-induced Decline in Ca2+i
The decline in fluo-3 fluorescence that accompanies the exposure of a dark-adapted rod to the laser spot is consistent with the fall in Ca2+i known to take place during the photoresponse (Yau and Nakatani, 1985; McNaughton et al., 1986; Ratto et al., 1988; Gray-Keller and Detwiler, 1994; McCarthy et al., 1994). The fluorescence signal was best fitted by the sum of two exponential components of approximately equal amplitude but with time constants differing by a factor of nearly 10. These observations agree well with the results obtained previously by others, who have also found that the decline in Ca2+i can be best fitted by two or three exponential components (Gray-Keller and Detwiler, 1994; McCarthy et al., 1996). In contrast, the electrogenic current carried by Na+/Ca2+-K+ exchange can in many cases be fitted adequately by just a single exponential (Yau and Nakatani, 1985). However, this discrepancy between the time courses of the fall in Ca2+i and the decline in the rate of Ca2+ efflux through the exchanger may be more apparent than real. The fluorescent probe measures the change in Ca2+i spatially averaged over the volume of the outer segment encompassed by the laser spot, whose diameter approaches that of the outer segment. On the other hand, the exchange current is governed by Ca2+i near the plasma membrane. The difference in their time courses may reflect the restricted radial diffusion and slowly equilibrating low affinity buffering of Ca2+ (McCarthy et al., 1996). It is also possible that the multiple components seen in the decline in Ca2+i represent the presence of multiple buffers of differing affinity and capacity within the rod outer segment (Hodgkin et al., 1987; Lagnado et al., 1992).
Calibration of these light-dependent fluorescence signals by exposure of the outer segment to solutions designed to raise or lower Ca2+i to levels much greater than or much less than the Kd of fluo-3 indicated that Ca2+i falls from 670 nM in darkness to 30 nM during response saturation. These values are broadly consistent with those obtained previously using other Ca2+ probes (McNaughton et al., 1986; Ratto et al., 1988; Gray-Keller and Detwiler, 1994; McCarthy et al., 1994; Younger et al., 1996). Since there was some uncertainty in the determination of Fmax as a result of the difficulty in reliably obtaining complete saturation of fluo-3 fluorescence when exposing the outer segment to ionomycin and high Ca2+, it seems possible that the value for Ca2+i in darkness may have been overestimated somewhat in our study. In contrast, after complete suppression of the circulating current, fluo-3 fluorescence was consistently greater than Fmin determined in situ by superfusing the outer segment with ionomycin and low Ca2+. Despite the bleach-induced saturation of the response by the preceding laser pulses, exposure to this solution resulted in a substantial increase in circulating current, indicating that Ca2+i must have been lowered further from its already substantially reduced value after bleaching. This qualitative observation adds support to our measurement of a value for Ca2+i significantly greater than zero even when the circulating current was completely suppressed. This contrasts with the much lower level of Ca2+i that would be expected if the Na+/Ca2+-K+ exchanger were to attain thermodynamic equilibrium (Cervetto et al., 1989; Perry and McNaughton, 1993). Therefore, these results suggest either that the exchanger is inhibited before this equilibrium is attained (Schnetkamp et al., 1991a), or that under these conditions a residual Ca2+ influx, perhaps from the inner segment, may balance a slow exchange extrusion of Ca2+.
Even when the outer segment was exposed to low Ca2+ and ionomycin, considerable fluorescence could still be excited, despite negligible fluo-3 fluorescence under zero Ca2+ conditions in the cuvette (Fig. 1, inset). It seems unlikely that this pedestal of fluorescence was caused by compartmentalization of fluo-3 within the outer segment (such as in the disks), since incubation with fluo-3 AM at 4°C or incorporation of fluo-3-free acid, both of which would be expected to reduce dye compartmentalization, produced a similar residual fluorescence after complete suppression of the circulating current. It is also difficult to account for this pedestal in terms of fluorescence from incompletely hydrolyzed dye, since fluo-3 exhibits negligible fluorescence in its esterified form. Instead, it seems more probable that this component of the total fluorescence may have originated from nonspecific binding of fluo-3 within the outer segment, perhaps to membrane phospholipids (McLaughlin and Brown, 1981) or to components of the cytoskeleton. Broadly comparable effects have been seen in previous studies that used fura-2 (McCarthy et al., 1994) or indo-1 (Gray-Keller and Detwiler, 1994). The pedestal of fluorescence that we observe appears to resemble most closely the slowly quenched pool of fura-2 fluorescence (McCarthy et al., 1994), since we saw no sign of the artifactual increases in Ca2+i that have been reported with free indo-1 (Gray-Keller and Detwiler, 1994). We therefore believe that once this pedestal is excluded, the light- and Ca2+-dependent fluo-3 fluorescence that remains is likely to represent a "well-behaved" measure of Ca2+i, as was the case for the rapidly quenched pool of fura-2 fluorescence (McCarthy et al., 1994).
Changes in Ca2+i After Photopigment Bleaching
The primary goal of this study was to investigate changes in rod outer segment Ca2+i after photopigment bleaching. Our measurements show that fluo-3 fluorescence is significantly depressed after bleaching, but recovers to a level not significantly different from the original dark-adapted value after regeneration of the photopigment with 11-cis-retinal, which also restores circulating current, response kinetics, and sensitivity (Jones et al., 1989; Corson et al., 1990). These systematic changes in fluorescence intensity indicate that the bleach-induced suppression of circulating current is accompanied by a persistent decrease in Ca2+i, which is restored when the photopigment is regenerated. Since bleach-induced changes in photoresponse sensitivity and kinetics are also abolished if this fall in Ca2+i is prevented (Matthews et al., 1996c), our observations are consistent with the notion that the reduction in Ca2+i after bleaching is responsible for the adaptation that ensues, and that its restoration after the regeneration of the photopigment leads to the recovery of dark-adapted sensitivity.
Outer segment Ca2+i is believed to be controlled by the dynamic balance between Ca2+ influx through the light-sensitive channel (Yau and Nakatani, 1984; Hodgkin et al., 1985) and efflux via Na+/Ca2+-K+ exchange (Yau and Nakatani, 1985; Hodgkin et al., 1987; Lagnado et al., 1992). Partial suppression of the circulating current by steady light is known to cause a graded decrease in Ca2+i (Gray-Keller and Detwiler, 1994, 1996; Younger et al., 1996), which is believed to be largely, if not exclusively, responsible for light adaptation (Koutalos et al., 1995; Matthews, 1995, 1996; Koutalos and Yau, 1996). It is difficult in our experiments to make a quantitative comparison between the magnitude of the fall in Ca2+i and the degree of suppression of the circulating current by bleaching, since the laser spot excites fluorescence and bleaches pigment within only a small region of the outer segment, while the suction pipette collects a fixed fraction of the entire outer segment current. The suppression of circulating current is believed to be closely localized to the site of illumination or bleaching (Lamb et al., 1981; Baylor and Lamb, 1982; Cornwall et al., 1990); thus, it seems likely that the degree of current suppression in the area of the outer segment illuminated by the laser spot will have been rather greater than indicated by the suction pipette recording. Nonetheless, the partial suppression of both circulating current and fluo-3 fluorescence by bleaching and their near-complete restoration by 11-cis-retinal suggests that they are modulated in a parallel fashion during the photopigment cycle. This is consistent with the notion that the persistent activation of the transduction cascade that follows bleaching (Cornwall and Fain, 1994; Cornwall et al., 1995) leads, through partial suppression of the circulating current, to a reduction in Ca2+ influx and a corresponding fall in Ca2+i. This bleach-induced fall in Ca2+i will not only accelerate the rate of cGMP production by guanylyl cyclase (Koch and Stryer, 1988; Gorczyca et al., 1994), but also act on the cyclic nucleotide–gated channel (Hsu and Molday, 1993; Nakatani et al., 1995) and at other sites earlier in the transduction cascade (Kawamura and Murakami, 1991; Kawamura, 1993; Lagnado and Baylor, 1994) to produce the characteristic adaptational changes in sensitivity and response waveform that accompany bleaching in the isolated rod, in much the same way as does steady light.
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ACKNOWLEDGMENTS |
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This work was supported by grants EY-01844, EY-07026, and EY-01157 from the National Eye Institute of the National Institutes of Health, and by a grant from the Wellcome Trust.
Submitted: 2 September 1997
Accepted: 20 October 1997
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 G. L. Fain, H. R. Matthews, M. C. Cornwall, and Y. Koutalos Adaptation in Vertebrate Photoreceptors Physiol Rev, January 1, 2001; 81(1): 117 - 151. [Abstract] [Full Text] [PDF]
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C. F. M. Prinsen, R. T. Szerencsei, and P. P. M. Schnetkamp Molecular Cloning and Functional Expression of the Potassium-Dependent Sodium-Calcium Exchanger from Human and Chicken Retinal Cone Photoreceptors J. Neurosci., February 15, 2000; 20(4): 1424 - 1434. [Abstract] [Full Text] [PDF]
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 S. Tachibanaki, K. Nanda, K. Sasaki, K. Ozaki, and S. Kawamura Amino Acid Residues of S-modulin Responsible for Interaction with Rhodopsin Kinase J. Biol. Chem., February 4, 2000; 275(5): 3313 - 3319. [Abstract] [Full Text] [PDF]
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 K. Haug-Collet, B. Pearson, R. Webel, R.T. Szerencsei, R.J. Winkfein, P.P.M. Schnetkamp, and N.J. Colley Cloning and Characterization of a Potassium-Dependent Sodium/Calcium Exchanger in Drosophila J. Cell Biol., November 1, 1999; 147(3): 659 - 670. [Abstract] [Full Text] [PDF]
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E. V. Olshevskaya, A. N. Ermilov, and A. M. Dizhoor Dimerization of Guanylyl Cyclase-activating Protein and a Mechanism of Photoreceptor Guanylyl Cyclase Activation J. Biol. Chem., September 3, 1999; 274(36): 25583 - 25587. [Abstract] [Full Text] [PDF]
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J. B. Ames, A. M. Dizhoor, M. Ikura, K. Palczewski, and L. Stryer Three-dimensional Structure of Guanylyl Cyclase Activating Protein-2, a Calcium-sensitive Modulator of Photoreceptor Guanylyl Cyclases J. Biol. Chem., July 2, 1999; 274(27): 19329 - 19337. [Abstract] [Full Text] [PDF]
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 W. Denk and P. B. Detwiler Optical recording of light-evoked calcium signals in the functionally intact retina PNAS, June 8, 1999; 96(12): 7035 - 7040. [Abstract] [Full Text] [PDF]
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E. V. Olshevskaya, S. Boikov, A. Ermilov, D. Krylov, J. B. Hurley, and A. M. Dizhoor Mapping Functional Domains of the Guanylate Cyclase Regulator Protein, GCAP-2 J. Biol. Chem., April 16, 1999; 274(16): 10823 - 10832. [Abstract] [Full Text] [PDF]
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V. J. Kefalov, M. Carter Cornwall, and R. K. Crouch Occupancy of the Chromophore Binding Site of Opsin Activates Visual Transduction in Rod Photoreceptors J. Gen. Physiol., March 1, 1999; 113(3): 491 - 503. [Abstract] [Full Text] [PDF]
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A.P. Sampath, H.R. Matthews, M.C. Cornwall, J. Bandarchi, and G.L. Fain Light-dependent Changes in Outer Segment Free-Ca2+ Concentration in Salamander Cone Photoreceptors J. Gen. Physiol., February 1, 1999; 113(2): 267 - 277. [Abstract] [Full Text] [PDF]
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T. I. Rebrik and J. I. Korenbrot In Intact Cone Photoreceptors, a Ca2+-dependent, Diffusible Factor Modulates the cGMP-gated Ion Channels Differently than in Rods J. Gen. Physiol., November 1, 1998; 112(5): 537 - 548. [Abstract] [Full Text] [PDF]
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M. A. Erickson, L. Lagnado, S. Zozulya, T. A. Neubert, L. Stryer, and D. A. Baylor The effect of recombinant recoverin on the photoresponse of truncated rod photoreceptors PNAS, May 26, 1998; 95(11): 6474 - 6479. [Abstract] [Full Text] [PDF]
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