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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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WT ERC currents from fused giant cells. (A) Flashes (500 nm, 4.08 × 108 photons/μm2) elicited ERC currents that were extinguished by successive stimuli during the first bleach of this cell after primary chromophore regeneration. The integrated R2 charge motion (Qi) is shown to the right of each trace. The location of the ERC traces with respect to the ordinate is arbitrary to allow easy comparison. Note the prominent inward R1 current in the ERC stimulated by the first flash. In this cell, charge was extinguished by the third flash. (B) After a 10-min period of dark adaptation, a second series of 500-nm flashes was given and ERC charge motions had recovered. ERC signals were again extinguished by seven successive flashes. No R1 signals were elicited in the secondary extinctions. (C) An additional 10-min period of dark adaptation also promoted recovery of the ERC signal, here measured with 570-nm flashes (3.38 × 108 photons/μm2). (D) Kinetic data shows the normalized amount of ERC charge that recovers over time with dark adaptation following flash extinction (500 nm) of the signal (n = 2 cells). An exponential growth function was fit to the data: Q = a · [1 − exp(−b · t)]. From the fit a (0.86256 ± 0.06836 min−1) and b (0.31745 ± 0.10078 min−1) were determined (correlation = 0.96526, P < 0.001), where b is the rate of regeneration.
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Figure 1: WT ERC currents from fused giant cells. (A) Flashes (500 nm, 4.08 × 108 photons/μm2) elicited ERC currents that were extinguished by successive stimuli during the first bleach of this cell after primary chromophore regeneration. The integrated R2 charge motion (Qi) is shown to the right of each trace. The location of the ERC traces with respect to the ordinate is arbitrary to allow easy comparison. Note the prominent inward R1 current in the ERC stimulated by the first flash. In this cell, charge was extinguished by the third flash. (B) After a 10-min period of dark adaptation, a second series of 500-nm flashes was given and ERC charge motions had recovered. ERC signals were again extinguished by seven successive flashes. No R1 signals were elicited in the secondary extinctions. (C) An additional 10-min period of dark adaptation also promoted recovery of the ERC signal, here measured with 570-nm flashes (3.38 × 108 photons/μm2). (D) Kinetic data shows the normalized amount of ERC charge that recovers over time with dark adaptation following flash extinction (500 nm) of the signal (n = 2 cells). An exponential growth function was fit to the data: Q = a · [1 − exp(−b · t)]. From the fit a (0.86256 ± 0.06836 min−1) and b (0.31745 ± 0.10078 min−1) were determined (correlation = 0.96526, P < 0.001), where b is the rate of regeneration.

Mentions: Fig. 1 A shows a giant cell ERC obtained on the first flash series (500 nm) after a 30-min regeneration. Flash stimuli were given at 500 nm to extinguish the ERC signal into background whole-cell noise. Both the submillisecond negative R1 current and the millisecond-order larger, and positive R2 current were routinely observed and essentially identical to those recorded in photoreceptors (see Hestrin and Korenbrot 1990; Makino et al. 1991; Sullivan and Shukla 1999; Sullivan et al. 2000). While not detectable in single unfused cells, in giant cells the R1 phase of the ERC was recordable, apparently due to increased total rhodopsin and improved SNR. After subtraction of baseline current, integration of each ERC lead to the charge motion (Qi) in femtocoulombs attributable to each phase. Successive flashes progressively extinguished both phases of the ERC until no responses were observed above background noise. This was consistent with bleaching of plasma membrane rhodopsin due to photolysis. The observation that giant cells spontaneously recovered ERC signals after a complete bleach by simple dark adaptation for 10–15 min without added chromophore was quite surprising. Unless otherwise stated, cells were not exposed to additional 11cRet once the coverslip was removed from regeneration buffer and placed in E-1 buffer in the recording chamber. Subsequent flash photolysis after post-bleach dark adaptation lead to robust ERC signals that were again extinguished by additional flashes at 500 nm (Fig. 1 B). Another period of dark adaptation promoted spontaneous recovery of the ERC that was again extinguished by successive 570-nm flashes (Fig. 1 C). The R2 but not R1 signals were recorded after spontaneous regeneration of visual pigment that occurs during 10-min dark adaptation (see discussion).


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

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

WT ERC currents from fused giant cells. (A) Flashes (500 nm, 4.08 × 108 photons/μm2) elicited ERC currents that were extinguished by successive stimuli during the first bleach of this cell after primary chromophore regeneration. The integrated R2 charge motion (Qi) is shown to the right of each trace. The location of the ERC traces with respect to the ordinate is arbitrary to allow easy comparison. Note the prominent inward R1 current in the ERC stimulated by the first flash. In this cell, charge was extinguished by the third flash. (B) After a 10-min period of dark adaptation, a second series of 500-nm flashes was given and ERC charge motions had recovered. ERC signals were again extinguished by seven successive flashes. No R1 signals were elicited in the secondary extinctions. (C) An additional 10-min period of dark adaptation also promoted recovery of the ERC signal, here measured with 570-nm flashes (3.38 × 108 photons/μm2). (D) Kinetic data shows the normalized amount of ERC charge that recovers over time with dark adaptation following flash extinction (500 nm) of the signal (n = 2 cells). An exponential growth function was fit to the data: Q = a · [1 − exp(−b · t)]. From the fit a (0.86256 ± 0.06836 min−1) and b (0.31745 ± 0.10078 min−1) were determined (correlation = 0.96526, P < 0.001), where b is the rate of regeneration.
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Related In: Results  -  Collection

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Figure 1: WT ERC currents from fused giant cells. (A) Flashes (500 nm, 4.08 × 108 photons/μm2) elicited ERC currents that were extinguished by successive stimuli during the first bleach of this cell after primary chromophore regeneration. The integrated R2 charge motion (Qi) is shown to the right of each trace. The location of the ERC traces with respect to the ordinate is arbitrary to allow easy comparison. Note the prominent inward R1 current in the ERC stimulated by the first flash. In this cell, charge was extinguished by the third flash. (B) After a 10-min period of dark adaptation, a second series of 500-nm flashes was given and ERC charge motions had recovered. ERC signals were again extinguished by seven successive flashes. No R1 signals were elicited in the secondary extinctions. (C) An additional 10-min period of dark adaptation also promoted recovery of the ERC signal, here measured with 570-nm flashes (3.38 × 108 photons/μm2). (D) Kinetic data shows the normalized amount of ERC charge that recovers over time with dark adaptation following flash extinction (500 nm) of the signal (n = 2 cells). An exponential growth function was fit to the data: Q = a · [1 − exp(−b · t)]. From the fit a (0.86256 ± 0.06836 min−1) and b (0.31745 ± 0.10078 min−1) were determined (correlation = 0.96526, P < 0.001), where b is the rate of regeneration.
Mentions: Fig. 1 A shows a giant cell ERC obtained on the first flash series (500 nm) after a 30-min regeneration. Flash stimuli were given at 500 nm to extinguish the ERC signal into background whole-cell noise. Both the submillisecond negative R1 current and the millisecond-order larger, and positive R2 current were routinely observed and essentially identical to those recorded in photoreceptors (see Hestrin and Korenbrot 1990; Makino et al. 1991; Sullivan and Shukla 1999; Sullivan et al. 2000). While not detectable in single unfused cells, in giant cells the R1 phase of the ERC was recordable, apparently due to increased total rhodopsin and improved SNR. After subtraction of baseline current, integration of each ERC lead to the charge motion (Qi) in femtocoulombs attributable to each phase. Successive flashes progressively extinguished both phases of the ERC until no responses were observed above background noise. This was consistent with bleaching of plasma membrane rhodopsin due to photolysis. The observation that giant cells spontaneously recovered ERC signals after a complete bleach by simple dark adaptation for 10–15 min without added chromophore was quite surprising. Unless otherwise stated, cells were not exposed to additional 11cRet once the coverslip was removed from regeneration buffer and placed in E-1 buffer in the recording chamber. Subsequent flash photolysis after post-bleach dark adaptation lead to robust ERC signals that were again extinguished by additional flashes at 500 nm (Fig. 1 B). Another period of dark adaptation promoted spontaneous recovery of the ERC that was again extinguished by successive 570-nm flashes (Fig. 1 C). The R2 but not R1 signals were recorded after spontaneous regeneration of visual pigment that occurs during 10-min dark adaptation (see discussion).

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