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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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9-cis-Retinal regenerates ERC signals. Cells were exposed to 25 μM 9cRet complexed to 2% FAF-BSA in regeneration buffer for over 30 min. (Left) ERC signals from a cell that was photolyzed with 500-nm flashes after the primary regeneration are small and extinguish with successive flashes. (Middle) After 10 min of dark adaptation, spontaneous recovery of ERCs was found with similar R2 kinetics compared with those found after primary extinction. Similar results were found in two additional cells. Holding potential for two cells was +30 mV and for one cell was 0 mV. (Right) An ERC charge extinction analysis allowed Pt to be extracted from the single exponential fit for the secondary regeneration. The cell was held at 0 mV. Pt was 1.03 × 10−9 μm2.
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Figure 8: 9-cis-Retinal regenerates ERC signals. Cells were exposed to 25 μM 9cRet complexed to 2% FAF-BSA in regeneration buffer for over 30 min. (Left) ERC signals from a cell that was photolyzed with 500-nm flashes after the primary regeneration are small and extinguish with successive flashes. (Middle) After 10 min of dark adaptation, spontaneous recovery of ERCs was found with similar R2 kinetics compared with those found after primary extinction. Similar results were found in two additional cells. Holding potential for two cells was +30 mV and for one cell was 0 mV. (Right) An ERC charge extinction analysis allowed Pt to be extracted from the single exponential fit for the secondary regeneration. The cell was held at 0 mV. Pt was 1.03 × 10−9 μm2.

Mentions: Cells were regenerated with 9cRet to test the feasibility of ERC investigation of rhodopsin activation in analogue visual pigments. Analogue visual pigments are usually formed from WT opsin and a synthetic retinal known to have unique properties (e.g., to block Meta-II formation), but could also be formed from synthetic retinals and site-specific opsin mutants. The naturally occurring 9cRet analogue forms isorhodopsin, a stable ground state pigment that is generated in a photostationary state with rhodopsin and bathorhodopsin (Birge et al. 1988). Once isorhodopsin is photoactivated to bathorhodopsin, the same sequence of bleaching intermediates occur as compared with normal rhodopsin activation. ERCs were recorded in three of four fused cells regenerated in 9cRet and signals were uniformly small. This may in part be related to the cell sizes used [Cmem 85.7, 11.6 (probably a single cell), and 48.6 pF]. Fig. 8 shows primary (left) and secondary (middle) extinctions of ERCs with 500-nm flashes for a fused cell regenerated with 9cRet. ERC signals in fused WT-HEK293 cells regenerated with 9cRet were smaller than those regenerated with 11cRet in the population of cells studied. However, the ERC R2 waveform was similar. Pt was determined by flash series extinction at 500 nm (70 nm) for this cell and found to be 1.03 × 10−9 μm2, which is similar to the Pt we calculate for isorhodopsin at peak extinction at 483 nm (5.55 × 10−9 μm2). This value is arrived at by first calculating the molecular cross section (αλ) from the extinction coefficient for isorhodopsin at 483 nm (44,000 M−1 · cm−1) (αλ = 3.82 × 10−21 * ∈λ) (αλ = 1.68 × 10−8 μm2) and multiplying αλ by the quantal efficiency of isorhodopsin of 0.33 (Crouch et al. 1975). At the flash intensities used in these experiments (4.08 × 108 photons/μm2), ∼90% of isorhodopsin pigment molecules should absorb at least one photon, with ∼55% of these being odd-numbered isomerizations that would proceed forward to bleaching and ∼45% being even-numbered isomerizations that would result in photoconversion to ground state species. Thus, Pt could be suppressed by photoregeneration at the flash intensities used.


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

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

9-cis-Retinal regenerates ERC signals. Cells were exposed to 25 μM 9cRet complexed to 2% FAF-BSA in regeneration buffer for over 30 min. (Left) ERC signals from a cell that was photolyzed with 500-nm flashes after the primary regeneration are small and extinguish with successive flashes. (Middle) After 10 min of dark adaptation, spontaneous recovery of ERCs was found with similar R2 kinetics compared with those found after primary extinction. Similar results were found in two additional cells. Holding potential for two cells was +30 mV and for one cell was 0 mV. (Right) An ERC charge extinction analysis allowed Pt to be extracted from the single exponential fit for the secondary regeneration. The cell was held at 0 mV. Pt was 1.03 × 10−9 μm2.
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Related In: Results  -  Collection

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Figure 8: 9-cis-Retinal regenerates ERC signals. Cells were exposed to 25 μM 9cRet complexed to 2% FAF-BSA in regeneration buffer for over 30 min. (Left) ERC signals from a cell that was photolyzed with 500-nm flashes after the primary regeneration are small and extinguish with successive flashes. (Middle) After 10 min of dark adaptation, spontaneous recovery of ERCs was found with similar R2 kinetics compared with those found after primary extinction. Similar results were found in two additional cells. Holding potential for two cells was +30 mV and for one cell was 0 mV. (Right) An ERC charge extinction analysis allowed Pt to be extracted from the single exponential fit for the secondary regeneration. The cell was held at 0 mV. Pt was 1.03 × 10−9 μm2.
Mentions: Cells were regenerated with 9cRet to test the feasibility of ERC investigation of rhodopsin activation in analogue visual pigments. Analogue visual pigments are usually formed from WT opsin and a synthetic retinal known to have unique properties (e.g., to block Meta-II formation), but could also be formed from synthetic retinals and site-specific opsin mutants. The naturally occurring 9cRet analogue forms isorhodopsin, a stable ground state pigment that is generated in a photostationary state with rhodopsin and bathorhodopsin (Birge et al. 1988). Once isorhodopsin is photoactivated to bathorhodopsin, the same sequence of bleaching intermediates occur as compared with normal rhodopsin activation. ERCs were recorded in three of four fused cells regenerated in 9cRet and signals were uniformly small. This may in part be related to the cell sizes used [Cmem 85.7, 11.6 (probably a single cell), and 48.6 pF]. Fig. 8 shows primary (left) and secondary (middle) extinctions of ERCs with 500-nm flashes for a fused cell regenerated with 9cRet. ERC signals in fused WT-HEK293 cells regenerated with 9cRet were smaller than those regenerated with 11cRet in the population of cells studied. However, the ERC R2 waveform was similar. Pt was determined by flash series extinction at 500 nm (70 nm) for this cell and found to be 1.03 × 10−9 μm2, which is similar to the Pt we calculate for isorhodopsin at peak extinction at 483 nm (5.55 × 10−9 μm2). This value is arrived at by first calculating the molecular cross section (αλ) from the extinction coefficient for isorhodopsin at 483 nm (44,000 M−1 · cm−1) (αλ = 3.82 × 10−21 * ∈λ) (αλ = 1.68 × 10−8 μm2) and multiplying αλ by the quantal efficiency of isorhodopsin of 0.33 (Crouch et al. 1975). At the flash intensities used in these experiments (4.08 × 108 photons/μm2), ∼90% of isorhodopsin pigment molecules should absorb at least one photon, with ∼55% of these being odd-numbered isomerizations that would proceed forward to bleaching and ∼45% being even-numbered isomerizations that would result in photoconversion to ground state species. Thus, Pt could be suppressed by photoregeneration at the flash intensities used.

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