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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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Related in: MedlinePlus

Spectral sensitivity of the ERC response. ERC responses to single initial flashes at several wavelengths are shown. Data was collected from cells after spontaneous dark regenerations. Filters were 30 nm (FWHM) except for the 540-nm filter, which was 10 nm (FWHM) (see materials and methods). For each waveform, the current trace was multiplicatively scaled by the relative ratio of the flash intensities at the test wavelength relative to 500 nm. A double exponential function was fitted to the R2 relaxation of the 500-nm response. The same function was overlaid with the 440-, 480-, and 520-nm ERC responses and provided a good fit. Action spectra of the R2 ERC response (bottom right) is shown. For each cell (n = 3), a single flash was given at each wavelength with a 10-min period of dark adaptation between each flash. Single flashes were given so that cells would be less exhausted and regeneration would be more uniform over recording epochs at different wavelengths. The integrated R2 charge, Qi, was obtained at each wavelength. Qi values for each cell were multiplicatively scaled by the ratio of photon densities (relative to 500 nm) so that the ERC responses at equal numbers of photons were obtained at the respective wavelengths. R2 charges were normalized to the maximum charge for a cell to compensate for differences in fused cell rhodopsin content and allow statistical comparison of action spectra. The means (±SEM) are plotted versus wavelength. The variance about the mean at 500 nm is artifactually low because of the normalization procedure. In one cell, holding potential was +30 mV, while in the other two it was 0 mV. Spectra were not affected by this change in holding potential. The absorbance spectrum of human rhodopsin regenerated with 11cRet and purified from WT-HEK293 cells was normalized to the α band peak extinction at 493 nm and plotted over the normalized mean ERC action spectrum on the same abscissa.
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Figure 3: Spectral sensitivity of the ERC response. ERC responses to single initial flashes at several wavelengths are shown. Data was collected from cells after spontaneous dark regenerations. Filters were 30 nm (FWHM) except for the 540-nm filter, which was 10 nm (FWHM) (see materials and methods). For each waveform, the current trace was multiplicatively scaled by the relative ratio of the flash intensities at the test wavelength relative to 500 nm. A double exponential function was fitted to the R2 relaxation of the 500-nm response. The same function was overlaid with the 440-, 480-, and 520-nm ERC responses and provided a good fit. Action spectra of the R2 ERC response (bottom right) is shown. For each cell (n = 3), a single flash was given at each wavelength with a 10-min period of dark adaptation between each flash. Single flashes were given so that cells would be less exhausted and regeneration would be more uniform over recording epochs at different wavelengths. The integrated R2 charge, Qi, was obtained at each wavelength. Qi values for each cell were multiplicatively scaled by the ratio of photon densities (relative to 500 nm) so that the ERC responses at equal numbers of photons were obtained at the respective wavelengths. R2 charges were normalized to the maximum charge for a cell to compensate for differences in fused cell rhodopsin content and allow statistical comparison of action spectra. The means (±SEM) are plotted versus wavelength. The variance about the mean at 500 nm is artifactually low because of the normalization procedure. In one cell, holding potential was +30 mV, while in the other two it was 0 mV. Spectra were not affected by this change in holding potential. The absorbance spectrum of human rhodopsin regenerated with 11cRet and purified from WT-HEK293 cells was normalized to the α band peak extinction at 493 nm and plotted over the normalized mean ERC action spectrum on the same abscissa.

Mentions: Photosensitivity (Pt) is the product of quantal efficiency (γ) and the wavelength-dependent absorbance cross section (αλ). The absorbance cross section of wild-type human rhodopsin is 1.53 × 10−8 μm2 (calculated from an extinction coefficient of 40,000 M−1 cm−1 at 493 nm (Wald and Brown 1958; Dartnall 1968; Knowles and Dartnall 1977) and γ is 0.67, leading to a Pt of 10−8 μm2 for normal human rhodopsin at peak extinction (493 nm). Pt can be used to estimate the fraction of rhodopsin molecules absorbing at least one photon per flash using the zero-order term of the Poisson equation [1 − Po = 1 − exp(−Pt · i)], where i is the flash intensity (photons/μm2) and Po is the fraction that absorbs no photons [Poisson Eq.: Pn = (Pt*i)n*exp(−Pt*i)/n!, where n is the number of absorptions per chromophore]. In this calculation, one adjusts αλ by the ratio of absorbance at the wavelength of interest to that at peak extinction. γ is assumed to be constant and independent of wavelength. For the 70-nm bandpass filters used in these experiments (centered at 350, 430, 500, and 570 nm), the fraction of molecules absorbing at least one photon were estimated to be 0.159, 0.716, 0.963, and 0.273, respectively. For the 30- and 10-nm bandpass filters used in these experiments (centered at 400, 440, 480, 500, 520, 540, 580, and 620 nm), the fraction of rhodopsin molecules absorbing at least one photon were estimated at 0.226, 0.626, 0.831, 0.80, 0.733, 0.44, 0.122, and 0.013, respectively. These calculations assume no orientational factors, no self-screening effects, and transparent cellular media. Thus, microbeam flash intensities were not expected to be experimentally limiting for flash photolytic stimulation of expressed rhodopsin pigments, except perhaps for the 620-nm stimulus. The maximum extent of rhodopsin bleaching (i.e., formation of Meta-II) after a single flash is 50% (Williams 1965, Williams 1974) because of second (or even-numbered) photon reabsorptions during the lifetimes of early states that have high quantal efficiency to photochemically regenerate the ground state (e.g., bathorhodopsin, lumirhodopsin). Flashes at 400, 580, and 620 nm were likely to elicit only single photon absorptions (>90%). Flashes at other wavelengths (440, 480, 500, 520, and 540 nm) were more likely to include even-numbered absorptions (relative fraction of total for even numbered absorptions 0.31, 0.415, 0.405, 0.367, and 0.22, respectively). The absolute flash intensities (108 photons/μm2) at the various center wavelengths (parentheses) used in action spectra experiments were as follows: 1.20 (400 nm), 2.29 (440 nm), 2.27 (480 nm), 1.96 (500 nm), 2.02 (520 nm), 1.49 (540 nm), 2.49 (580 nm), and 1.68 (620 nm). The relative ratios of absolute flash intensities relative to that at 500 nm were 0.61, 1.16, 1.15, 1.0, 1.03, 0.76, 1.27, and 0.85, respectively. To scale charge motions for action spectra (see Fig. 3), the reciprocal of these scale factors were used to multiplicatively scale the integrated charge motions.


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

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

Spectral sensitivity of the ERC response. ERC responses to single initial flashes at several wavelengths are shown. Data was collected from cells after spontaneous dark regenerations. Filters were 30 nm (FWHM) except for the 540-nm filter, which was 10 nm (FWHM) (see materials and methods). For each waveform, the current trace was multiplicatively scaled by the relative ratio of the flash intensities at the test wavelength relative to 500 nm. A double exponential function was fitted to the R2 relaxation of the 500-nm response. The same function was overlaid with the 440-, 480-, and 520-nm ERC responses and provided a good fit. Action spectra of the R2 ERC response (bottom right) is shown. For each cell (n = 3), a single flash was given at each wavelength with a 10-min period of dark adaptation between each flash. Single flashes were given so that cells would be less exhausted and regeneration would be more uniform over recording epochs at different wavelengths. The integrated R2 charge, Qi, was obtained at each wavelength. Qi values for each cell were multiplicatively scaled by the ratio of photon densities (relative to 500 nm) so that the ERC responses at equal numbers of photons were obtained at the respective wavelengths. R2 charges were normalized to the maximum charge for a cell to compensate for differences in fused cell rhodopsin content and allow statistical comparison of action spectra. The means (±SEM) are plotted versus wavelength. The variance about the mean at 500 nm is artifactually low because of the normalization procedure. In one cell, holding potential was +30 mV, while in the other two it was 0 mV. Spectra were not affected by this change in holding potential. The absorbance spectrum of human rhodopsin regenerated with 11cRet and purified from WT-HEK293 cells was normalized to the α band peak extinction at 493 nm and plotted over the normalized mean ERC action spectrum on the same abscissa.
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Related In: Results  -  Collection

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Figure 3: Spectral sensitivity of the ERC response. ERC responses to single initial flashes at several wavelengths are shown. Data was collected from cells after spontaneous dark regenerations. Filters were 30 nm (FWHM) except for the 540-nm filter, which was 10 nm (FWHM) (see materials and methods). For each waveform, the current trace was multiplicatively scaled by the relative ratio of the flash intensities at the test wavelength relative to 500 nm. A double exponential function was fitted to the R2 relaxation of the 500-nm response. The same function was overlaid with the 440-, 480-, and 520-nm ERC responses and provided a good fit. Action spectra of the R2 ERC response (bottom right) is shown. For each cell (n = 3), a single flash was given at each wavelength with a 10-min period of dark adaptation between each flash. Single flashes were given so that cells would be less exhausted and regeneration would be more uniform over recording epochs at different wavelengths. The integrated R2 charge, Qi, was obtained at each wavelength. Qi values for each cell were multiplicatively scaled by the ratio of photon densities (relative to 500 nm) so that the ERC responses at equal numbers of photons were obtained at the respective wavelengths. R2 charges were normalized to the maximum charge for a cell to compensate for differences in fused cell rhodopsin content and allow statistical comparison of action spectra. The means (±SEM) are plotted versus wavelength. The variance about the mean at 500 nm is artifactually low because of the normalization procedure. In one cell, holding potential was +30 mV, while in the other two it was 0 mV. Spectra were not affected by this change in holding potential. The absorbance spectrum of human rhodopsin regenerated with 11cRet and purified from WT-HEK293 cells was normalized to the α band peak extinction at 493 nm and plotted over the normalized mean ERC action spectrum on the same abscissa.
Mentions: Photosensitivity (Pt) is the product of quantal efficiency (γ) and the wavelength-dependent absorbance cross section (αλ). The absorbance cross section of wild-type human rhodopsin is 1.53 × 10−8 μm2 (calculated from an extinction coefficient of 40,000 M−1 cm−1 at 493 nm (Wald and Brown 1958; Dartnall 1968; Knowles and Dartnall 1977) and γ is 0.67, leading to a Pt of 10−8 μm2 for normal human rhodopsin at peak extinction (493 nm). Pt can be used to estimate the fraction of rhodopsin molecules absorbing at least one photon per flash using the zero-order term of the Poisson equation [1 − Po = 1 − exp(−Pt · i)], where i is the flash intensity (photons/μm2) and Po is the fraction that absorbs no photons [Poisson Eq.: Pn = (Pt*i)n*exp(−Pt*i)/n!, where n is the number of absorptions per chromophore]. In this calculation, one adjusts αλ by the ratio of absorbance at the wavelength of interest to that at peak extinction. γ is assumed to be constant and independent of wavelength. For the 70-nm bandpass filters used in these experiments (centered at 350, 430, 500, and 570 nm), the fraction of molecules absorbing at least one photon were estimated to be 0.159, 0.716, 0.963, and 0.273, respectively. For the 30- and 10-nm bandpass filters used in these experiments (centered at 400, 440, 480, 500, 520, 540, 580, and 620 nm), the fraction of rhodopsin molecules absorbing at least one photon were estimated at 0.226, 0.626, 0.831, 0.80, 0.733, 0.44, 0.122, and 0.013, respectively. These calculations assume no orientational factors, no self-screening effects, and transparent cellular media. Thus, microbeam flash intensities were not expected to be experimentally limiting for flash photolytic stimulation of expressed rhodopsin pigments, except perhaps for the 620-nm stimulus. The maximum extent of rhodopsin bleaching (i.e., formation of Meta-II) after a single flash is 50% (Williams 1965, Williams 1974) because of second (or even-numbered) photon reabsorptions during the lifetimes of early states that have high quantal efficiency to photochemically regenerate the ground state (e.g., bathorhodopsin, lumirhodopsin). Flashes at 400, 580, and 620 nm were likely to elicit only single photon absorptions (>90%). Flashes at other wavelengths (440, 480, 500, 520, and 540 nm) were more likely to include even-numbered absorptions (relative fraction of total for even numbered absorptions 0.31, 0.415, 0.405, 0.367, and 0.22, respectively). The absolute flash intensities (108 photons/μm2) at the various center wavelengths (parentheses) used in action spectra experiments were as follows: 1.20 (400 nm), 2.29 (440 nm), 2.27 (480 nm), 1.96 (500 nm), 2.02 (520 nm), 1.49 (540 nm), 2.49 (580 nm), and 1.68 (620 nm). The relative ratios of absolute flash intensities relative to that at 500 nm were 0.61, 1.16, 1.15, 1.0, 1.03, 0.76, 1.27, and 0.85, respectively. To scale charge motions for action spectra (see Fig. 3), the reciprocal of these scale factors were used to multiplicatively scale the integrated charge motions.

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