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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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Comparison of ERC signals in WT, D83N, and E134Q human rhodopsins. The first ERC response to a 500-nm flash stimulus after primary regeneration (left) and after secondary recovery (right) is shown for WT human, D83N, and E134Q rod rhodopsin. In comparison with the WT pigment, D83N and E134Q lack R1 signals on the primary extinction series. D83N has a simpler R2 kinetic relaxation and loses the stretched exponential characteristic seen in the WT ERC, whereas the E134Q ERC is a simple outward current of short duration that is well fit by a single exponential. Attempts were made to fit single, double, or triple exponential models to secondary ERC R2 relaxations. If each exponential corresponds to a unique rate leading from a particular electrical state (Markovian), the WT and D83N R2 relaxations are reliably fit by the sum of two exponentials, consistent with three unique electrical states, whereas the single exponential fitting of the E134Q R2 relaxation suggests a two-state model. Residuals are shown beneath each fitted R2 waveform. R2 relaxations were fit with double (A, B > 0) or single exponential models (B = 0) of the form: ERCt=A·e−t−t0τa+B·e−t−t0τb, where τa and τb and A and B are the time constants and weights of the fitted exponentials, and to is the time at which fitting was initiated (just after the peak of R2).
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Figure 9: Comparison of ERC signals in WT, D83N, and E134Q human rhodopsins. The first ERC response to a 500-nm flash stimulus after primary regeneration (left) and after secondary recovery (right) is shown for WT human, D83N, and E134Q rod rhodopsin. In comparison with the WT pigment, D83N and E134Q lack R1 signals on the primary extinction series. D83N has a simpler R2 kinetic relaxation and loses the stretched exponential characteristic seen in the WT ERC, whereas the E134Q ERC is a simple outward current of short duration that is well fit by a single exponential. Attempts were made to fit single, double, or triple exponential models to secondary ERC R2 relaxations. If each exponential corresponds to a unique rate leading from a particular electrical state (Markovian), the WT and D83N R2 relaxations are reliably fit by the sum of two exponentials, consistent with three unique electrical states, whereas the single exponential fitting of the E134Q R2 relaxation suggests a two-state model. Residuals are shown beneath each fitted R2 waveform. R2 relaxations were fit with double (A, B > 0) or single exponential models (B = 0) of the form: ERCt=A·e−t−t0τa+B·e−t−t0τb, where τa and τb and A and B are the time constants and weights of the fitted exponentials, and to is the time at which fitting was initiated (just after the peak of R2).

Mentions: Fig. 9 shows ERC signals in response to the first 500-nm flash after primary regeneration and secondary recovery in fused giant cells containing WT, D83N, or E134Q human rhodopsins. The WT pigment generated strong R1 signals during the primary bleach. R1 signals are rarely seen during the secondary or subsequent extinctions indicating that, if present, the size is below the limits of detection at the flash intensities used. Large (>40 pA) WT ERC signals typically require two exponentials to fit the time course of the R2 relaxation over the first 100 ms. Residuals are shown beneath the ERC waveforms. R1 signals were not observed in D83N rhodopsin during primary extinction in cells with R2 charges of the same order as seen in fused WT cells that had R1 signals. Like WT, the D83N R2 relaxation typically requires two exponentials to reliably fit its relaxation. However, D83N signals appear to lack the “stretched” exponential appearance seen in many large WT signals during the 100 ms after the flash. The E134Q ERC signal was distinctly different from the WT signal. R1 signals were not observed during primary extinctions. Moreover, the outward R2 signals in E134Q rhodopsin-expressing cells were markedly simplified in comparison with WT or D83N ERCs. The relaxation was brief and required only a single exponential to fit its decay. Like WT ERCs, D83N and E134Q ERC signals extinguished with successive flashes and had spectral sensitivity consistent with pigments absorbing ∼500 nm (Sakmar et al. 1989; Nathans 1990).


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

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

Comparison of ERC signals in WT, D83N, and E134Q human rhodopsins. The first ERC response to a 500-nm flash stimulus after primary regeneration (left) and after secondary recovery (right) is shown for WT human, D83N, and E134Q rod rhodopsin. In comparison with the WT pigment, D83N and E134Q lack R1 signals on the primary extinction series. D83N has a simpler R2 kinetic relaxation and loses the stretched exponential characteristic seen in the WT ERC, whereas the E134Q ERC is a simple outward current of short duration that is well fit by a single exponential. Attempts were made to fit single, double, or triple exponential models to secondary ERC R2 relaxations. If each exponential corresponds to a unique rate leading from a particular electrical state (Markovian), the WT and D83N R2 relaxations are reliably fit by the sum of two exponentials, consistent with three unique electrical states, whereas the single exponential fitting of the E134Q R2 relaxation suggests a two-state model. Residuals are shown beneath each fitted R2 waveform. R2 relaxations were fit with double (A, B > 0) or single exponential models (B = 0) of the form: ERCt=A·e−t−t0τa+B·e−t−t0τb, where τa and τb and A and B are the time constants and weights of the fitted exponentials, and to is the time at which fitting was initiated (just after the peak of R2).
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Related In: Results  -  Collection

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Figure 9: Comparison of ERC signals in WT, D83N, and E134Q human rhodopsins. The first ERC response to a 500-nm flash stimulus after primary regeneration (left) and after secondary recovery (right) is shown for WT human, D83N, and E134Q rod rhodopsin. In comparison with the WT pigment, D83N and E134Q lack R1 signals on the primary extinction series. D83N has a simpler R2 kinetic relaxation and loses the stretched exponential characteristic seen in the WT ERC, whereas the E134Q ERC is a simple outward current of short duration that is well fit by a single exponential. Attempts were made to fit single, double, or triple exponential models to secondary ERC R2 relaxations. If each exponential corresponds to a unique rate leading from a particular electrical state (Markovian), the WT and D83N R2 relaxations are reliably fit by the sum of two exponentials, consistent with three unique electrical states, whereas the single exponential fitting of the E134Q R2 relaxation suggests a two-state model. Residuals are shown beneath each fitted R2 waveform. R2 relaxations were fit with double (A, B > 0) or single exponential models (B = 0) of the form: ERCt=A·e−t−t0τa+B·e−t−t0τb, where τa and τb and A and B are the time constants and weights of the fitted exponentials, and to is the time at which fitting was initiated (just after the peak of R2).
Mentions: Fig. 9 shows ERC signals in response to the first 500-nm flash after primary regeneration and secondary recovery in fused giant cells containing WT, D83N, or E134Q human rhodopsins. The WT pigment generated strong R1 signals during the primary bleach. R1 signals are rarely seen during the secondary or subsequent extinctions indicating that, if present, the size is below the limits of detection at the flash intensities used. Large (>40 pA) WT ERC signals typically require two exponentials to fit the time course of the R2 relaxation over the first 100 ms. Residuals are shown beneath the ERC waveforms. R1 signals were not observed in D83N rhodopsin during primary extinction in cells with R2 charges of the same order as seen in fused WT cells that had R1 signals. Like WT, the D83N R2 relaxation typically requires two exponentials to reliably fit its relaxation. However, D83N signals appear to lack the “stretched” exponential appearance seen in many large WT signals during the 100 ms after the flash. The E134Q ERC signal was distinctly different from the WT signal. R1 signals were not observed during primary extinctions. Moreover, the outward R2 signals in E134Q rhodopsin-expressing cells were markedly simplified in comparison with WT or D83N ERCs. The relaxation was brief and required only a single exponential to fit its decay. Like WT ERCs, D83N and E134Q ERC signals extinguished with successive flashes and had spectral sensitivity consistent with pigments absorbing ∼500 nm (Sakmar et al. 1989; Nathans 1990).

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