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Normal and mutant rhodopsin activation measured with the early receptor current in a unicellular expression system.

Shukla P, Sullivan JM - J. Gen. Physiol. (1999)

Bottom Line: After signal extinction, dark adaptation without added 11-cis-retinal resulted in spontaneous pigment regeneration from an intracellular store of chromophore remaining from earlier loading.These results indicate that the ERC can be photoregenerated from the metarhodopsin-II state.D83N ERCs were simplified in comparison with normal rhodopsin, while E134Q ERCs had only the early phase of charge motion.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, State University of New York, Health Science Center at Syracuse, Syracuse, New York 13210, USA.

ABSTRACT
The early receptor current (ERC) represents molecular charge movement during rhodopsin conformational dynamics. To determine whether this time-resolved assay can probe various aspects of structure-function relationships in rhodopsin, we first measured properties of expressed normal human rhodopsin with ERC recordings. These studies were conducted in single fused giant cells containing on the order of a picogram of regenerated pigment. The action spectrum of the ERC of normal human opsin regenerated with 11-cis-retinal was fit by the human rhodopsin absorbance spectrum. Successive flashes extinguished ERC signals consistent with bleaching of a rhodopsin photopigment with a normal range of photosensitivity. ERC signals followed the univariance principle since millisecond-order relaxation kinetics were independent of the wavelength of the flash stimulus. After signal extinction, dark adaptation without added 11-cis-retinal resulted in spontaneous pigment regeneration from an intracellular store of chromophore remaining from earlier loading. After the ERC was extinguished, 350-nm flashes overlapping metarhodopsin-II absorption promoted immediate recovery of ERC charge motions identified by subsequent 500-nm flashes. Small inverted R(2) signals were seen in response to some 350-nm flashes. These results indicate that the ERC can be photoregenerated from the metarhodopsin-II state. Regeneration with 9-cis-retinal permits recording of ERC signals consistent with flash activation of isorhodopsin. We initiated structure-function studies by measuring ERC signals in cells expressing the D83N and E134Q mutant human rhodopsin pigments. D83N ERCs were simplified in comparison with normal rhodopsin, while E134Q ERCs had only the early phase of charge motion. This study demonstrates that properties of normal rhodopsin can be accurately measured with the ERC assay and that a structure-function investigation of rapid activation processes in analogue and mutant visual pigments is feasible in a live unicellular environment.

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Photoregeneration from Meta-II380. (A) 500-nm flashes (4.08 × 108 photons/μm2) were used to extinguish the ERC signal into the whole-cell current noise. Immediately thereafter, near UV flashes (350 nm, 8.07 × 107 photons/μm2) overlapping Meta-II absorption (peak 380 nm) were given. In this experiment, either 5 (left), 10 (middle), or 20 (right) UV flashes were given after the initial extinction, which required 27, 67, and 182 s, respectively, to deliver. 500-nm flashes were given immediately after the UV flashes to determine how much ground state ERC charge was recovered. In each panel, the ERC response to the first and last 500-nm flashes that initially extinguished the signal are shown (top and middle, respectively) (secondary regeneration). (Bottom) The ERC in response to first flash at 500 nm immediately after the set of UV flashes is shown. (B) The amount of recovered charge after UV flashes was normalized to the total amount of charge measured in the extinction immediately before the respective set of UV flashes to control for the increase in total R2 charge in this cell over time. Percent regeneration is plotted versus cumulative UV flash dose for two experiments. In the first experiment, 5, 10, or 20 UV flashes were given (▴) (data shown in A). Approximately 65% of the charge was recovered after five flashes with additional charge recovered by 10 and 20 flashes. In another experiment (▪), one to five UV flashes were given over 12, 7, 11, 19, and 22 s, respectively. Approximately 55% of the ERC charge was recovered by the third flash and there was little change after three subsequent flashes. These data were fit by a saturating Boltzmann function with a saturation point (A2) of 0.575, a nodal point (I1/2) of 108 photons/μm2, and a sensitivity factor (dI) of 0.19 × 108 photons/μm2) (below). (C) Inverted ERC signals with a slower time course compared with positive R2 were seen in response to 350-nm flashes. ERC signals are shown for a single representative cell (61.9 μm, Cmem = 126.2 pF) in response to 500- and 350-nm flashes (secondary regeneration). After the 500-nm flashes had extinguished ERC R2 signals, 350-nm flashes were given. The upper trace is the ERC response to a 500-nm flash and the lower trace is the response to a 350-nm flash on the same time scale. Without constraint, third order polynomials fit the inward going charge motion. Note an apparent reversed R1 phase in the response of the larger cell: Q = (A1 − A2)/{1 + exp[(I − I1/2)/dI]} + A2 (A1 = 0).
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Figure 7: Photoregeneration from Meta-II380. (A) 500-nm flashes (4.08 × 108 photons/μm2) were used to extinguish the ERC signal into the whole-cell current noise. Immediately thereafter, near UV flashes (350 nm, 8.07 × 107 photons/μm2) overlapping Meta-II absorption (peak 380 nm) were given. In this experiment, either 5 (left), 10 (middle), or 20 (right) UV flashes were given after the initial extinction, which required 27, 67, and 182 s, respectively, to deliver. 500-nm flashes were given immediately after the UV flashes to determine how much ground state ERC charge was recovered. In each panel, the ERC response to the first and last 500-nm flashes that initially extinguished the signal are shown (top and middle, respectively) (secondary regeneration). (Bottom) The ERC in response to first flash at 500 nm immediately after the set of UV flashes is shown. (B) The amount of recovered charge after UV flashes was normalized to the total amount of charge measured in the extinction immediately before the respective set of UV flashes to control for the increase in total R2 charge in this cell over time. Percent regeneration is plotted versus cumulative UV flash dose for two experiments. In the first experiment, 5, 10, or 20 UV flashes were given (▴) (data shown in A). Approximately 65% of the charge was recovered after five flashes with additional charge recovered by 10 and 20 flashes. In another experiment (▪), one to five UV flashes were given over 12, 7, 11, 19, and 22 s, respectively. Approximately 55% of the ERC charge was recovered by the third flash and there was little change after three subsequent flashes. These data were fit by a saturating Boltzmann function with a saturation point (A2) of 0.575, a nodal point (I1/2) of 108 photons/μm2, and a sensitivity factor (dI) of 0.19 × 108 photons/μm2) (below). (C) Inverted ERC signals with a slower time course compared with positive R2 were seen in response to 350-nm flashes. ERC signals are shown for a single representative cell (61.9 μm, Cmem = 126.2 pF) in response to 500- and 350-nm flashes (secondary regeneration). After the 500-nm flashes had extinguished ERC R2 signals, 350-nm flashes were given. The upper trace is the ERC response to a 500-nm flash and the lower trace is the response to a 350-nm flash on the same time scale. Without constraint, third order polynomials fit the inward going charge motion. Note an apparent reversed R1 phase in the response of the larger cell: Q = (A1 − A2)/{1 + exp[(I − I1/2)/dI]} + A2 (A1 = 0).

Mentions: Previous studies have shown that the ground state of rhodopsin can be photoregenerated from Meta-II380 by near UV flashes delivered concurrent with its lifetime (Williams 1964, Williams 1968; Williams and Breil 1968; Cafiso and Hubbell 1980; Drachev et al. 1981; Arnis and Hofmann 1995). Photolysis of Meta-II generated inverted ERP signals (Cone and Cobbs 1969; Ebrey 1968; Cafiso and Hubbell 1980; Drachev et al. 1981). To investigate whether photoregeneration could be assayed with ERC measurements, 500-nm flashes were used to extinguish the ERC into background cell noise, whereupon 350-nm flashes (70 nm FWHM) overlapping the Meta-II absorption (350 nm, 8.02 × 107 photons/μm2) were delivered immediately and in rapid succession. The number of near UV flashes (1–5, 5, 10, and 20) delivered was varied to affect an increasing dose of photoregeneration stimulus to the Meta-II remaining in the cell. Immediately after the near UV flashes were delivered, additional flashes at 500 nm were used to measure the level of regeneration as the ERC R2 charge. Under the conditions of these experiments, the expected lifetime of Meta-II at room temperature in a membrane environment is on the order of several minutes (van Breugel et al., 1979; Parkes and Liebman 1984). Fig. 7 A shows ERC responses from an experiment where 5, 10, or 20 350-nm flashes were given to promote photoregeneration of ground state rhodopsin from Meta-II. The top trace in each case is the ERC response to the first 500-nm flash in the series used to initially extinguish the signal. The middle shows the response to the last 500-nm flash indicating the level of extinction into the whole-cell current noise. The bottom shows the first flash at 500 nm after 5 (left), 10 (middle), or 20 (right) UV flashes. As the number of UV flashes is increased, the size of the recovered 500-nm ERC signal becomes similar to that found before the UV flashes. The ERC R2 current waveform in response to 500-nm flashes after the UV flashes is comparable in size and kinetics to the 500-nm signal that preceded bleaching whether 5, 10, or 20 UV stimuli were delivered. This data suggested that the ground state of the pigment (11cRet) is regenerated during the photoconversion process. In this cell, the amount of ERC R2 charge increased over the time period of successive extinctions. The underlying mechanism is not yet clear (see discussion). Fig. 7 B shows the percent of ERC charge recovered after 5 (65%), 10 (74%), or 20 (87%) UV flashes, measured against the UV flash dose. The period of time needed to deliver 5, 10, or 20 UV flashes was 27, 67, or 182 s, respectively, in this experiment. Since the kinetics of spontaneous regeneration has a time constant of ∼3 min (Fig. 1 D), ERC responses after 10 or 20 UV flashes could be significantly affected by chemical regeneration with chromophore rather than photoregeneration. Therefore, the charge recovery in response to one to five UV flashes was examined (Fig. 7 B). By the third UV flash, the process of regeneration had stabilized (∼55%) at the UV intensity used and no significant additional charge recovery was found when two additional UV flashes were given. The amount of charge regenerated by three to five UV flashes was similar to that which regenerated after five flashes of the same intensity in the experiment described above. Only 11 s were required to deliver the three UV flashes, making it highly unlikely that any significant chemical regeneration had contributed to the 55% charge recovery. Thus, UV stimuli promoted a large fractional recovery of R2 charge in a time frame that is much more rapid than the kinetics of spontaneous ERC regeneration by dark adaptation. These results strongly suggest that the ERC can be photoregenerated by additional photons overlapping the Meta-II bandwidth. The quantal efficiency appears favorable to studying the mechanism in greater detail.


Normal and mutant rhodopsin activation measured with the early receptor current in a unicellular expression system.

Shukla P, Sullivan JM - J. Gen. Physiol. (1999)

Photoregeneration from Meta-II380. (A) 500-nm flashes (4.08 × 108 photons/μm2) were used to extinguish the ERC signal into the whole-cell current noise. Immediately thereafter, near UV flashes (350 nm, 8.07 × 107 photons/μm2) overlapping Meta-II absorption (peak 380 nm) were given. In this experiment, either 5 (left), 10 (middle), or 20 (right) UV flashes were given after the initial extinction, which required 27, 67, and 182 s, respectively, to deliver. 500-nm flashes were given immediately after the UV flashes to determine how much ground state ERC charge was recovered. In each panel, the ERC response to the first and last 500-nm flashes that initially extinguished the signal are shown (top and middle, respectively) (secondary regeneration). (Bottom) The ERC in response to first flash at 500 nm immediately after the set of UV flashes is shown. (B) The amount of recovered charge after UV flashes was normalized to the total amount of charge measured in the extinction immediately before the respective set of UV flashes to control for the increase in total R2 charge in this cell over time. Percent regeneration is plotted versus cumulative UV flash dose for two experiments. In the first experiment, 5, 10, or 20 UV flashes were given (▴) (data shown in A). Approximately 65% of the charge was recovered after five flashes with additional charge recovered by 10 and 20 flashes. In another experiment (▪), one to five UV flashes were given over 12, 7, 11, 19, and 22 s, respectively. Approximately 55% of the ERC charge was recovered by the third flash and there was little change after three subsequent flashes. These data were fit by a saturating Boltzmann function with a saturation point (A2) of 0.575, a nodal point (I1/2) of 108 photons/μm2, and a sensitivity factor (dI) of 0.19 × 108 photons/μm2) (below). (C) Inverted ERC signals with a slower time course compared with positive R2 were seen in response to 350-nm flashes. ERC signals are shown for a single representative cell (61.9 μm, Cmem = 126.2 pF) in response to 500- and 350-nm flashes (secondary regeneration). After the 500-nm flashes had extinguished ERC R2 signals, 350-nm flashes were given. The upper trace is the ERC response to a 500-nm flash and the lower trace is the response to a 350-nm flash on the same time scale. Without constraint, third order polynomials fit the inward going charge motion. Note an apparent reversed R1 phase in the response of the larger cell: Q = (A1 − A2)/{1 + exp[(I − I1/2)/dI]} + A2 (A1 = 0).
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Figure 7: Photoregeneration from Meta-II380. (A) 500-nm flashes (4.08 × 108 photons/μm2) were used to extinguish the ERC signal into the whole-cell current noise. Immediately thereafter, near UV flashes (350 nm, 8.07 × 107 photons/μm2) overlapping Meta-II absorption (peak 380 nm) were given. In this experiment, either 5 (left), 10 (middle), or 20 (right) UV flashes were given after the initial extinction, which required 27, 67, and 182 s, respectively, to deliver. 500-nm flashes were given immediately after the UV flashes to determine how much ground state ERC charge was recovered. In each panel, the ERC response to the first and last 500-nm flashes that initially extinguished the signal are shown (top and middle, respectively) (secondary regeneration). (Bottom) The ERC in response to first flash at 500 nm immediately after the set of UV flashes is shown. (B) The amount of recovered charge after UV flashes was normalized to the total amount of charge measured in the extinction immediately before the respective set of UV flashes to control for the increase in total R2 charge in this cell over time. Percent regeneration is plotted versus cumulative UV flash dose for two experiments. In the first experiment, 5, 10, or 20 UV flashes were given (▴) (data shown in A). Approximately 65% of the charge was recovered after five flashes with additional charge recovered by 10 and 20 flashes. In another experiment (▪), one to five UV flashes were given over 12, 7, 11, 19, and 22 s, respectively. Approximately 55% of the ERC charge was recovered by the third flash and there was little change after three subsequent flashes. These data were fit by a saturating Boltzmann function with a saturation point (A2) of 0.575, a nodal point (I1/2) of 108 photons/μm2, and a sensitivity factor (dI) of 0.19 × 108 photons/μm2) (below). (C) Inverted ERC signals with a slower time course compared with positive R2 were seen in response to 350-nm flashes. ERC signals are shown for a single representative cell (61.9 μm, Cmem = 126.2 pF) in response to 500- and 350-nm flashes (secondary regeneration). After the 500-nm flashes had extinguished ERC R2 signals, 350-nm flashes were given. The upper trace is the ERC response to a 500-nm flash and the lower trace is the response to a 350-nm flash on the same time scale. Without constraint, third order polynomials fit the inward going charge motion. Note an apparent reversed R1 phase in the response of the larger cell: Q = (A1 − A2)/{1 + exp[(I − I1/2)/dI]} + A2 (A1 = 0).
Mentions: Previous studies have shown that the ground state of rhodopsin can be photoregenerated from Meta-II380 by near UV flashes delivered concurrent with its lifetime (Williams 1964, Williams 1968; Williams and Breil 1968; Cafiso and Hubbell 1980; Drachev et al. 1981; Arnis and Hofmann 1995). Photolysis of Meta-II generated inverted ERP signals (Cone and Cobbs 1969; Ebrey 1968; Cafiso and Hubbell 1980; Drachev et al. 1981). To investigate whether photoregeneration could be assayed with ERC measurements, 500-nm flashes were used to extinguish the ERC into background cell noise, whereupon 350-nm flashes (70 nm FWHM) overlapping the Meta-II absorption (350 nm, 8.02 × 107 photons/μm2) were delivered immediately and in rapid succession. The number of near UV flashes (1–5, 5, 10, and 20) delivered was varied to affect an increasing dose of photoregeneration stimulus to the Meta-II remaining in the cell. Immediately after the near UV flashes were delivered, additional flashes at 500 nm were used to measure the level of regeneration as the ERC R2 charge. Under the conditions of these experiments, the expected lifetime of Meta-II at room temperature in a membrane environment is on the order of several minutes (van Breugel et al., 1979; Parkes and Liebman 1984). Fig. 7 A shows ERC responses from an experiment where 5, 10, or 20 350-nm flashes were given to promote photoregeneration of ground state rhodopsin from Meta-II. The top trace in each case is the ERC response to the first 500-nm flash in the series used to initially extinguish the signal. The middle shows the response to the last 500-nm flash indicating the level of extinction into the whole-cell current noise. The bottom shows the first flash at 500 nm after 5 (left), 10 (middle), or 20 (right) UV flashes. As the number of UV flashes is increased, the size of the recovered 500-nm ERC signal becomes similar to that found before the UV flashes. The ERC R2 current waveform in response to 500-nm flashes after the UV flashes is comparable in size and kinetics to the 500-nm signal that preceded bleaching whether 5, 10, or 20 UV stimuli were delivered. This data suggested that the ground state of the pigment (11cRet) is regenerated during the photoconversion process. In this cell, the amount of ERC R2 charge increased over the time period of successive extinctions. The underlying mechanism is not yet clear (see discussion). Fig. 7 B shows the percent of ERC charge recovered after 5 (65%), 10 (74%), or 20 (87%) UV flashes, measured against the UV flash dose. The period of time needed to deliver 5, 10, or 20 UV flashes was 27, 67, or 182 s, respectively, in this experiment. Since the kinetics of spontaneous regeneration has a time constant of ∼3 min (Fig. 1 D), ERC responses after 10 or 20 UV flashes could be significantly affected by chemical regeneration with chromophore rather than photoregeneration. Therefore, the charge recovery in response to one to five UV flashes was examined (Fig. 7 B). By the third UV flash, the process of regeneration had stabilized (∼55%) at the UV intensity used and no significant additional charge recovery was found when two additional UV flashes were given. The amount of charge regenerated by three to five UV flashes was similar to that which regenerated after five flashes of the same intensity in the experiment described above. Only 11 s were required to deliver the three UV flashes, making it highly unlikely that any significant chemical regeneration had contributed to the 55% charge recovery. Thus, UV stimuli promoted a large fractional recovery of R2 charge in a time frame that is much more rapid than the kinetics of spontaneous ERC regeneration by dark adaptation. These results strongly suggest that the ERC can be photoregenerated by additional photons overlapping the Meta-II bandwidth. The quantal efficiency appears favorable to studying the mechanism in greater detail.

Bottom Line: After signal extinction, dark adaptation without added 11-cis-retinal resulted in spontaneous pigment regeneration from an intracellular store of chromophore remaining from earlier loading.These results indicate that the ERC can be photoregenerated from the metarhodopsin-II state.D83N ERCs were simplified in comparison with normal rhodopsin, while E134Q ERCs had only the early phase of charge motion.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, State University of New York, Health Science Center at Syracuse, Syracuse, New York 13210, USA.

ABSTRACT
The early receptor current (ERC) represents molecular charge movement during rhodopsin conformational dynamics. To determine whether this time-resolved assay can probe various aspects of structure-function relationships in rhodopsin, we first measured properties of expressed normal human rhodopsin with ERC recordings. These studies were conducted in single fused giant cells containing on the order of a picogram of regenerated pigment. The action spectrum of the ERC of normal human opsin regenerated with 11-cis-retinal was fit by the human rhodopsin absorbance spectrum. Successive flashes extinguished ERC signals consistent with bleaching of a rhodopsin photopigment with a normal range of photosensitivity. ERC signals followed the univariance principle since millisecond-order relaxation kinetics were independent of the wavelength of the flash stimulus. After signal extinction, dark adaptation without added 11-cis-retinal resulted in spontaneous pigment regeneration from an intracellular store of chromophore remaining from earlier loading. After the ERC was extinguished, 350-nm flashes overlapping metarhodopsin-II absorption promoted immediate recovery of ERC charge motions identified by subsequent 500-nm flashes. Small inverted R(2) signals were seen in response to some 350-nm flashes. These results indicate that the ERC can be photoregenerated from the metarhodopsin-II state. Regeneration with 9-cis-retinal permits recording of ERC signals consistent with flash activation of isorhodopsin. We initiated structure-function studies by measuring ERC signals in cells expressing the D83N and E134Q mutant human rhodopsin pigments. D83N ERCs were simplified in comparison with normal rhodopsin, while E134Q ERCs had only the early phase of charge motion. This study demonstrates that properties of normal rhodopsin can be accurately measured with the ERC assay and that a structure-function investigation of rapid activation processes in analogue and mutant visual pigments is feasible in a live unicellular environment.

Show MeSH
Related in: MedlinePlus