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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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R2 state complexity in WT, D83N, and E134Q rhodopsins. An ensemble of all time constants from exponential fitting (τa,b) was used to construct histograms for WT (A), D83N (B), and E134Q (C) rhodopsin R2 relaxations. The intracellular pH of the WT and D83N studies was 6.5, whereas in the E134Q studies it was 6.0, 6.5, 7.0, 7.5, or 8.0. Sums of Gaussian distributions functions were fitted to the histograms of time constants. WT rhodopsin requires at least three Gaussians, D83N requires two, and E134Q requires a single Gaussian to reliably represent the time constant histograms. Individual Gaussians are drawn over each peak and the composite Gaussian (sum) traces the envelope of represented density. Time constants and errors from the fitting are shown in Table .
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Figure 10: R2 state complexity in WT, D83N, and E134Q rhodopsins. An ensemble of all time constants from exponential fitting (τa,b) was used to construct histograms for WT (A), D83N (B), and E134Q (C) rhodopsin R2 relaxations. The intracellular pH of the WT and D83N studies was 6.5, whereas in the E134Q studies it was 6.0, 6.5, 7.0, 7.5, or 8.0. Sums of Gaussian distributions functions were fitted to the histograms of time constants. WT rhodopsin requires at least three Gaussians, D83N requires two, and E134Q requires a single Gaussian to reliably represent the time constant histograms. Individual Gaussians are drawn over each peak and the composite Gaussian (sum) traces the envelope of represented density. Time constants and errors from the fitting are shown in Table .

Mentions: To begin to characterize R2 relaxation, single or double exponential functions were fit to a large number of WT, D83N, and E134Q R2 signals from many cells of similar size range. Time constants associated with R2 relaxation, but not R1, are essentially independent of Cmem (Sullivan and Shukla 1999). Time constants were extracted from the first and second exponential terms (τa, τb). Since the identification of the respective components is dependent upon their weighting, it is possible that the τb constant could be assigned to the τa data set if the weighting of the τa component is small or unreliable (e.g., lower SNR). Therefore, all the time constants obtained for R2 relaxation were placed into a total ensemble, and histograms were generated from these populations for the WT, D83N, and E134Q datasets (Fig. 10, A–C). The WT pigment demonstrated a broad skewed distribution suggestive of density around three time constant ranges, whereas the E134Q distribution was simple and symmetrical and the D83N distribution was intermediate. To quantitatively characterize the ensemble of time constants, Gaussian distribution functions were fit to each histogram. The WT histogram was reliably fit by a sum of three gaussian distributions, centered at 4.1, 12.5, and 26.4 ms (Table ). There were also residuals with time constants longer than the three fitted distributions. This analysis demonstrates the kinetic complexity of the WT R2 relaxation and supported the conclusion of a minimum of three charge states with distinct lifetimes (see discussion). The D83N histogram was reliably fit to the sum of two Gaussian functions centered at 3.7 and 10.7 ms, still leaving some residuals. These time constants are comparable with the two fastest time constants measured in the first 100 ms of WT R2 relaxations, whereas the third τ found in WT appears to be missing (Table ). The E134Q histogram was distinctly different from both WT and D83N, requiring only a single Gaussian function with a peak centered at 4.4 ms. This single peak overlaps with the fastest time constant seen in the WT and D83N pigments (Table ). This work establishes an initial approach to parameterize the R2 relaxation. The intent is to use this approach as a means to quantify differences between WT and mutant ERC kinetics during the biochemically important time period of the R2 signal.


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

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

R2 state complexity in WT, D83N, and E134Q rhodopsins. An ensemble of all time constants from exponential fitting (τa,b) was used to construct histograms for WT (A), D83N (B), and E134Q (C) rhodopsin R2 relaxations. The intracellular pH of the WT and D83N studies was 6.5, whereas in the E134Q studies it was 6.0, 6.5, 7.0, 7.5, or 8.0. Sums of Gaussian distributions functions were fitted to the histograms of time constants. WT rhodopsin requires at least three Gaussians, D83N requires two, and E134Q requires a single Gaussian to reliably represent the time constant histograms. Individual Gaussians are drawn over each peak and the composite Gaussian (sum) traces the envelope of represented density. Time constants and errors from the fitting are shown in Table .
© Copyright Policy
Related In: Results  -  Collection

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Figure 10: R2 state complexity in WT, D83N, and E134Q rhodopsins. An ensemble of all time constants from exponential fitting (τa,b) was used to construct histograms for WT (A), D83N (B), and E134Q (C) rhodopsin R2 relaxations. The intracellular pH of the WT and D83N studies was 6.5, whereas in the E134Q studies it was 6.0, 6.5, 7.0, 7.5, or 8.0. Sums of Gaussian distributions functions were fitted to the histograms of time constants. WT rhodopsin requires at least three Gaussians, D83N requires two, and E134Q requires a single Gaussian to reliably represent the time constant histograms. Individual Gaussians are drawn over each peak and the composite Gaussian (sum) traces the envelope of represented density. Time constants and errors from the fitting are shown in Table .
Mentions: To begin to characterize R2 relaxation, single or double exponential functions were fit to a large number of WT, D83N, and E134Q R2 signals from many cells of similar size range. Time constants associated with R2 relaxation, but not R1, are essentially independent of Cmem (Sullivan and Shukla 1999). Time constants were extracted from the first and second exponential terms (τa, τb). Since the identification of the respective components is dependent upon their weighting, it is possible that the τb constant could be assigned to the τa data set if the weighting of the τa component is small or unreliable (e.g., lower SNR). Therefore, all the time constants obtained for R2 relaxation were placed into a total ensemble, and histograms were generated from these populations for the WT, D83N, and E134Q datasets (Fig. 10, A–C). The WT pigment demonstrated a broad skewed distribution suggestive of density around three time constant ranges, whereas the E134Q distribution was simple and symmetrical and the D83N distribution was intermediate. To quantitatively characterize the ensemble of time constants, Gaussian distribution functions were fit to each histogram. The WT histogram was reliably fit by a sum of three gaussian distributions, centered at 4.1, 12.5, and 26.4 ms (Table ). There were also residuals with time constants longer than the three fitted distributions. This analysis demonstrates the kinetic complexity of the WT R2 relaxation and supported the conclusion of a minimum of three charge states with distinct lifetimes (see discussion). The D83N histogram was reliably fit to the sum of two Gaussian functions centered at 3.7 and 10.7 ms, still leaving some residuals. These time constants are comparable with the two fastest time constants measured in the first 100 ms of WT R2 relaxations, whereas the third τ found in WT appears to be missing (Table ). The E134Q histogram was distinctly different from both WT and D83N, requiring only a single Gaussian function with a peak centered at 4.4 ms. This single peak overlaps with the fastest time constant seen in the WT and D83N pigments (Table ). This work establishes an initial approach to parameterize the R2 relaxation. The intent is to use this approach as a means to quantify differences between WT and mutant ERC kinetics during the biochemically important time period of the R2 signal.

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