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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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Tests of ERC linearity and independence. ERCs were recorded using flashes of different intensity (2.9 × 108 and 3.5 × 108 photons/μm2) at 500 nm in a spontaneously regenerated cell held at 0 mV. Responses were normalized to the peak of R2, and the responses were overlaid. The kinetics of R2 relaxation appear otherwise identical regardless of flash stimulus intensity. A graph of the charge motion versus flash intensity at 500 nm is also shown and is fit by a line with slope of 420.62 fC/108 photons per μm2 (R = 0.99039, P < 0.000138). The A and B marks above the line reflect the ERC responses shown.
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Figure 5: Tests of ERC linearity and independence. ERCs were recorded using flashes of different intensity (2.9 × 108 and 3.5 × 108 photons/μm2) at 500 nm in a spontaneously regenerated cell held at 0 mV. Responses were normalized to the peak of R2, and the responses were overlaid. The kinetics of R2 relaxation appear otherwise identical regardless of flash stimulus intensity. A graph of the charge motion versus flash intensity at 500 nm is also shown and is fit by a line with slope of 420.62 fC/108 photons per μm2 (R = 0.99039, P < 0.000138). The A and B marks above the line reflect the ERC responses shown.

Mentions: Rhodopsin regenerated in fused giant cells was activated by an intense flash microbeam apparatus described in detail elsewhere (Sullivan 1998). In brief, light from a xenon flash tube is collimated, filtered, and condensed into a 1-mm-core fused silica fiber optics for transmission to the epifluorescent port of the microscope (Diaphot; Nikon Inc.). The objective lens is used to condense the fiber output into a microbeam spot parafocal with the specimen plane where the giant cell is situated. The spot-size diameter [full-width-half-maximum (FWHM)] in these experiments is 228 μm, which is about three times the size of the largest giant cell used. In routine flash photolysis, three-cavity bandpass filter elements (350, 430, 500, and 570 nm) were used that had 70-nm bandpass (FWHM) centered on peak transmission wavelength. To acquire action spectra data, 30 nm FWHM bandpass filters (centered at 400, 440, 480, 500, 520, 580, and 620 nm, and a 540-nm filter with a 10 nm FWHM bandwidth) were used. The throughputs of all filters, except those at 350 (70) and 400 (30) nm, do not overlap with the absorption spectra of free chromophore (peak ≈ 374 nm) such that isomerization (cis → trans or trans → cis) of any free chromophore is not expected during flashes used to elicit ERCs. Unless otherwise mentioned, flashes were delivered at the maximum capacity of the instrument. Intensities were 108–109 photons/μm2 across the near UV/visible band. Flash microbeam intensities were measured using a calibrated photodiode placed over the specimen plane of the microscope. To regulate flash intensity output (see Fig. 5), the voltage on the flash tube energy storage capacitor was adjusted. Flash duration was only ∼14 μs, insuring that the Meta-I ⇔ Meta-II equilibrium (milliseconds) generated at room temperature in these experiments was not perturbed by photoregeneration to other states (Sullivan 1998). Flash duration did overlap with lifetimes of bathorhodopsin, blue-shifted intermediate, and lumirhodopsin such that photoregeneration from these states is possible. Since we were largely concerned with the millisecond-order R2 phase of the ERC, any photoregeneration from early bleaching intermediates should not perturb the charge motions occurring during R2, which correlate with the time scale of the Meta-I ⇔ Meta-II transition. Shielding and fiber optic transmission prevent contamination of the patch-clamp electronics with flash-associated noise.


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

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

Tests of ERC linearity and independence. ERCs were recorded using flashes of different intensity (2.9 × 108 and 3.5 × 108 photons/μm2) at 500 nm in a spontaneously regenerated cell held at 0 mV. Responses were normalized to the peak of R2, and the responses were overlaid. The kinetics of R2 relaxation appear otherwise identical regardless of flash stimulus intensity. A graph of the charge motion versus flash intensity at 500 nm is also shown and is fit by a line with slope of 420.62 fC/108 photons per μm2 (R = 0.99039, P < 0.000138). The A and B marks above the line reflect the ERC responses shown.
© Copyright Policy
Related In: Results  -  Collection

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Figure 5: Tests of ERC linearity and independence. ERCs were recorded using flashes of different intensity (2.9 × 108 and 3.5 × 108 photons/μm2) at 500 nm in a spontaneously regenerated cell held at 0 mV. Responses were normalized to the peak of R2, and the responses were overlaid. The kinetics of R2 relaxation appear otherwise identical regardless of flash stimulus intensity. A graph of the charge motion versus flash intensity at 500 nm is also shown and is fit by a line with slope of 420.62 fC/108 photons per μm2 (R = 0.99039, P < 0.000138). The A and B marks above the line reflect the ERC responses shown.
Mentions: Rhodopsin regenerated in fused giant cells was activated by an intense flash microbeam apparatus described in detail elsewhere (Sullivan 1998). In brief, light from a xenon flash tube is collimated, filtered, and condensed into a 1-mm-core fused silica fiber optics for transmission to the epifluorescent port of the microscope (Diaphot; Nikon Inc.). The objective lens is used to condense the fiber output into a microbeam spot parafocal with the specimen plane where the giant cell is situated. The spot-size diameter [full-width-half-maximum (FWHM)] in these experiments is 228 μm, which is about three times the size of the largest giant cell used. In routine flash photolysis, three-cavity bandpass filter elements (350, 430, 500, and 570 nm) were used that had 70-nm bandpass (FWHM) centered on peak transmission wavelength. To acquire action spectra data, 30 nm FWHM bandpass filters (centered at 400, 440, 480, 500, 520, 580, and 620 nm, and a 540-nm filter with a 10 nm FWHM bandwidth) were used. The throughputs of all filters, except those at 350 (70) and 400 (30) nm, do not overlap with the absorption spectra of free chromophore (peak ≈ 374 nm) such that isomerization (cis → trans or trans → cis) of any free chromophore is not expected during flashes used to elicit ERCs. Unless otherwise mentioned, flashes were delivered at the maximum capacity of the instrument. Intensities were 108–109 photons/μm2 across the near UV/visible band. Flash microbeam intensities were measured using a calibrated photodiode placed over the specimen plane of the microscope. To regulate flash intensity output (see Fig. 5), the voltage on the flash tube energy storage capacitor was adjusted. Flash duration was only ∼14 μs, insuring that the Meta-I ⇔ Meta-II equilibrium (milliseconds) generated at room temperature in these experiments was not perturbed by photoregeneration to other states (Sullivan 1998). Flash duration did overlap with lifetimes of bathorhodopsin, blue-shifted intermediate, and lumirhodopsin such that photoregeneration from these states is possible. Since we were largely concerned with the millisecond-order R2 phase of the ERC, any photoregeneration from early bleaching intermediates should not perturb the charge motions occurring during R2, which correlate with the time scale of the Meta-I ⇔ Meta-II transition. Shielding and fiber optic transmission prevent contamination of the patch-clamp electronics with flash-associated noise.

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