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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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Test of the univariance principle. The first ERC responses to flashes at 430, 500, and 570 nm in a single fused giant cell regenerated with 11cRet are shown. Responses are from a cell that had spontaneously regenerated and was held at +30 mV. Stimuli were generated with 70-nm bandpass filters and intensities were: 430 nm (3.63 × 108 photons/μm2), 500 nm (4.08 × 108 photons/μm2) and 570 nm (3.38 × 108 photons/μm2). ERCs at each stimulation wavelength were smoothed by adjacent point averaging (8), and then normalized to the smoothed peak of the R2 current such that the normalization was not to the noise band. The ERC R2 signal resulting from the 500-nm flash was then fit with a double exponential template. This template was then overlaid with the R2 signal from the 430- and 570-nm responses and provided a good fit at the other stimulation wavelengths (see also Fig. 3).
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Figure 6: Test of the univariance principle. The first ERC responses to flashes at 430, 500, and 570 nm in a single fused giant cell regenerated with 11cRet are shown. Responses are from a cell that had spontaneously regenerated and was held at +30 mV. Stimuli were generated with 70-nm bandpass filters and intensities were: 430 nm (3.63 × 108 photons/μm2), 500 nm (4.08 × 108 photons/μm2) and 570 nm (3.38 × 108 photons/μm2). ERCs at each stimulation wavelength were smoothed by adjacent point averaging (8), and then normalized to the smoothed peak of the R2 current such that the normalization was not to the noise band. The ERC R2 signal resulting from the 500-nm flash was then fit with a double exponential template. This template was then overlaid with the R2 signal from the 430- and 570-nm responses and provided a good fit at the other stimulation wavelengths (see also Fig. 3).

Mentions: According to the univariance principle, the energy of the photon (wavelength) should not affect the activation kinetics of independent rhodopsin molecules. Photon energy only affects the probability of absorption because the molecular cross section is a function of wavelength. Fig. 6 shows ERCs acquired from a giant cell at 430, 500, and 570 nm. ERC waveforms were normalized to the smoothed peak of the R2 current for comparison. A double exponential curve was generated to fit the relaxation kinetics of the R2 signal from the 500-nm stimulus. This template was then overlaid with the R2 signals from the 430- and 570-nm responses. The 500-nm template fits the R2 relaxations of the 430- and 570-nm responses rather well, even though the SNR was lower with 570-nm stimuli because the absolute response was smaller. Although the amplitudes of the ERCs and total charge motion of the R2 signal vary with wavelength, the kinetics of R2 relaxation are similar. Similarly, the large 500-nm R2 response in Fig. 3 was fit with a double exponential function, and this template was overlaid with the large 440-, 480-, and 520-nm ERC responses and provided a good fit. The 400- and 540-nm ERC responses were of lower SNR and were not fit well by the template. ERC data in Fig. 3 was collected with 30 nm FWHM stimuli. In the rhodopsin expression system, photon energy does not affect the kinetics of the state transitions in rhodopsin, which is consistent with the univariance principle.


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

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

Test of the univariance principle. The first ERC responses to flashes at 430, 500, and 570 nm in a single fused giant cell regenerated with 11cRet are shown. Responses are from a cell that had spontaneously regenerated and was held at +30 mV. Stimuli were generated with 70-nm bandpass filters and intensities were: 430 nm (3.63 × 108 photons/μm2), 500 nm (4.08 × 108 photons/μm2) and 570 nm (3.38 × 108 photons/μm2). ERCs at each stimulation wavelength were smoothed by adjacent point averaging (8), and then normalized to the smoothed peak of the R2 current such that the normalization was not to the noise band. The ERC R2 signal resulting from the 500-nm flash was then fit with a double exponential template. This template was then overlaid with the R2 signal from the 430- and 570-nm responses and provided a good fit at the other stimulation wavelengths (see also Fig. 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230543&req=5

Figure 6: Test of the univariance principle. The first ERC responses to flashes at 430, 500, and 570 nm in a single fused giant cell regenerated with 11cRet are shown. Responses are from a cell that had spontaneously regenerated and was held at +30 mV. Stimuli were generated with 70-nm bandpass filters and intensities were: 430 nm (3.63 × 108 photons/μm2), 500 nm (4.08 × 108 photons/μm2) and 570 nm (3.38 × 108 photons/μm2). ERCs at each stimulation wavelength were smoothed by adjacent point averaging (8), and then normalized to the smoothed peak of the R2 current such that the normalization was not to the noise band. The ERC R2 signal resulting from the 500-nm flash was then fit with a double exponential template. This template was then overlaid with the R2 signal from the 430- and 570-nm responses and provided a good fit at the other stimulation wavelengths (see also Fig. 3).
Mentions: According to the univariance principle, the energy of the photon (wavelength) should not affect the activation kinetics of independent rhodopsin molecules. Photon energy only affects the probability of absorption because the molecular cross section is a function of wavelength. Fig. 6 shows ERCs acquired from a giant cell at 430, 500, and 570 nm. ERC waveforms were normalized to the smoothed peak of the R2 current for comparison. A double exponential curve was generated to fit the relaxation kinetics of the R2 signal from the 500-nm stimulus. This template was then overlaid with the R2 signals from the 430- and 570-nm responses. The 500-nm template fits the R2 relaxations of the 430- and 570-nm responses rather well, even though the SNR was lower with 570-nm stimuli because the absolute response was smaller. Although the amplitudes of the ERCs and total charge motion of the R2 signal vary with wavelength, the kinetics of R2 relaxation are similar. Similarly, the large 500-nm R2 response in Fig. 3 was fit with a double exponential function, and this template was overlaid with the large 440-, 480-, and 520-nm ERC responses and provided a good fit. The 400- and 540-nm ERC responses were of lower SNR and were not fit well by the template. ERC data in Fig. 3 was collected with 30 nm FWHM stimuli. In the rhodopsin expression system, photon energy does not affect the kinetics of the state transitions in rhodopsin, which is consistent with the univariance principle.

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