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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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ERC exhaustion photosensitivity is consistent with rhodopsin bleaching. ERC currents (not shown) from a giant cell were recorded in response to flashes at 570 nm (70). Once the 570-nm flashes no longer generated ERC signals above noise, more effective 500-nm flashes were given that activated the remaining unbleached rhodopsin in the cell. The cell was held at +30 mV. R2 charges were obtained by integrating each ERC signal and plotted versus cumulative photon density for a single bleach. 570-nm flashes promoted progressive loss of ERC charge and 500-nm flashes were able to promote further extinction of ERC charge after the 570-nm flashes were no longer effective. Exhaustion curves at the two wavelengths were fit independently to . (B) The logarithmic transform of the Qi values was plotted on a linear cumulative intensity scale and lines were fit to both 570- and 500-nm components of the overall R2 charge extinction. (C) A collage of ERC exhaustion trials for a single large giant cell held at 0 mV is shown. The primary extinction (▪) and three later secondary extinctions at 500 nm (•, ▴, and ▾) are shown as well as single extinctions at 570 (×) and 430 (♦) nm. In all cases, the flashes promoted a progressive decrease in ERC charge with successive flash number at constant stimulus intensity (500 nm: 4.08 × 108, 430 nm: 3.62 × 108, and 570 nm: 3.38 × 108 photons/μm2). The three extinctions at 500 nm resulted in similar exhaustion curves and Pt estimates. (D) By fitting a single exponential decay model () to data from many experiments (n = 7 cells, 24 extinctions), Pt was determined. The means (±SEM) of Pt are plotted for primary bleaches (1), secondary bleaches (2), and at different wavelengths generated by 70-nm bandwidth filters [500(70), 430(70), and 570(70)] or 30-nm bandwidth filters [500(30)]. Data were collected from five cells held at 0-mV holding potential and two cells held at +30 mV. Means for Pt were not different on the basis of parametric (one-way analysis of variance) and nonparametric (Kruskal-Wallis analysis of variance) tests.
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Figure 4: ERC exhaustion photosensitivity is consistent with rhodopsin bleaching. ERC currents (not shown) from a giant cell were recorded in response to flashes at 570 nm (70). Once the 570-nm flashes no longer generated ERC signals above noise, more effective 500-nm flashes were given that activated the remaining unbleached rhodopsin in the cell. The cell was held at +30 mV. R2 charges were obtained by integrating each ERC signal and plotted versus cumulative photon density for a single bleach. 570-nm flashes promoted progressive loss of ERC charge and 500-nm flashes were able to promote further extinction of ERC charge after the 570-nm flashes were no longer effective. Exhaustion curves at the two wavelengths were fit independently to . (B) The logarithmic transform of the Qi values was plotted on a linear cumulative intensity scale and lines were fit to both 570- and 500-nm components of the overall R2 charge extinction. (C) A collage of ERC exhaustion trials for a single large giant cell held at 0 mV is shown. The primary extinction (▪) and three later secondary extinctions at 500 nm (•, ▴, and ▾) are shown as well as single extinctions at 570 (×) and 430 (♦) nm. In all cases, the flashes promoted a progressive decrease in ERC charge with successive flash number at constant stimulus intensity (500 nm: 4.08 × 108, 430 nm: 3.62 × 108, and 570 nm: 3.38 × 108 photons/μm2). The three extinctions at 500 nm resulted in similar exhaustion curves and Pt estimates. (D) By fitting a single exponential decay model () to data from many experiments (n = 7 cells, 24 extinctions), Pt was determined. The means (±SEM) of Pt are plotted for primary bleaches (1), secondary bleaches (2), and at different wavelengths generated by 70-nm bandwidth filters [500(70), 430(70), and 570(70)] or 30-nm bandwidth filters [500(30)]. Data were collected from five cells held at 0-mV holding potential and two cells held at +30 mV. Means for Pt were not different on the basis of parametric (one-way analysis of variance) and nonparametric (Kruskal-Wallis analysis of variance) tests.

Mentions: Successive flashes at a single wavelength and intensity promoted progressive loss of ERC R2 charge until no further signal was obtained above background current noise. Since the interstimulus intervals between successive flashes were only ∼10 s, pigment regeneration was minimal between stimuli, and regeneration should not contribute to the extinction progression. The spectral sensitivity of the ERC governs not only the efficacy of successive bleaches, but also single bleaches. Fig. 4 A shows the Qi extinction of R2 in response to successive flashes at 570 and then 500 nm in a single giant cell. Cumulative flash intensity delivered is used as the dependent variable. After ERC charge was effectively extinguished into noise by 570-nm flashes, 500-nm flashes of greater effective intensity were immediately delivered. The additional extinguishable ERC charge found with 500-nm flashes indicated residual ground state rhodopsin in the cell after the 570-nm flashes. This resulted because 570-nm flashes are not as effective at eliciting ERC currents as are flashes at 500 nm given the relative absorbance of WT human rhodopsin at 570 vs. 493 nm (peak absorbance) (OD570/OD493 = 0.436) (Wald and Brown 1958). Similar findings occur when bleaching at other wavelengths is followed by flash photolysis at 500 nm. The relative probability of activating rhodopsin, taken as the ratio of absorbance cross sections, is only ∼12% at 570 nm relative to 500 nm (α570/α493 = 0.115), assuming equal photon density at the two wavelengths. At the maximal flash strengths used, the fraction of rhodopsin molecules absorbing at least one photon at 500 vs. 570 nm was estimated to be ∼0.96 and 0.27, respectively. Thus, the flash system does not deliver sufficient photons at 570 nm to compensate for the lower probability of activation. This illustrates that detection of rhodopsin charge motions depends on the unitary charge motion, which should be the same at any wavelength (see univariance below), and the number of activated rhodopsin molecules that mobilize charge and sum into an ensemble ERC current. Even at peak wavelength (≈500 nm), a given flash intensity may not be sufficient to generate ERC currents above noise because the ensemble ERC current lies within the noise band.


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

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

ERC exhaustion photosensitivity is consistent with rhodopsin bleaching. ERC currents (not shown) from a giant cell were recorded in response to flashes at 570 nm (70). Once the 570-nm flashes no longer generated ERC signals above noise, more effective 500-nm flashes were given that activated the remaining unbleached rhodopsin in the cell. The cell was held at +30 mV. R2 charges were obtained by integrating each ERC signal and plotted versus cumulative photon density for a single bleach. 570-nm flashes promoted progressive loss of ERC charge and 500-nm flashes were able to promote further extinction of ERC charge after the 570-nm flashes were no longer effective. Exhaustion curves at the two wavelengths were fit independently to . (B) The logarithmic transform of the Qi values was plotted on a linear cumulative intensity scale and lines were fit to both 570- and 500-nm components of the overall R2 charge extinction. (C) A collage of ERC exhaustion trials for a single large giant cell held at 0 mV is shown. The primary extinction (▪) and three later secondary extinctions at 500 nm (•, ▴, and ▾) are shown as well as single extinctions at 570 (×) and 430 (♦) nm. In all cases, the flashes promoted a progressive decrease in ERC charge with successive flash number at constant stimulus intensity (500 nm: 4.08 × 108, 430 nm: 3.62 × 108, and 570 nm: 3.38 × 108 photons/μm2). The three extinctions at 500 nm resulted in similar exhaustion curves and Pt estimates. (D) By fitting a single exponential decay model () to data from many experiments (n = 7 cells, 24 extinctions), Pt was determined. The means (±SEM) of Pt are plotted for primary bleaches (1), secondary bleaches (2), and at different wavelengths generated by 70-nm bandwidth filters [500(70), 430(70), and 570(70)] or 30-nm bandwidth filters [500(30)]. Data were collected from five cells held at 0-mV holding potential and two cells held at +30 mV. Means for Pt were not different on the basis of parametric (one-way analysis of variance) and nonparametric (Kruskal-Wallis analysis of variance) tests.
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Figure 4: ERC exhaustion photosensitivity is consistent with rhodopsin bleaching. ERC currents (not shown) from a giant cell were recorded in response to flashes at 570 nm (70). Once the 570-nm flashes no longer generated ERC signals above noise, more effective 500-nm flashes were given that activated the remaining unbleached rhodopsin in the cell. The cell was held at +30 mV. R2 charges were obtained by integrating each ERC signal and plotted versus cumulative photon density for a single bleach. 570-nm flashes promoted progressive loss of ERC charge and 500-nm flashes were able to promote further extinction of ERC charge after the 570-nm flashes were no longer effective. Exhaustion curves at the two wavelengths were fit independently to . (B) The logarithmic transform of the Qi values was plotted on a linear cumulative intensity scale and lines were fit to both 570- and 500-nm components of the overall R2 charge extinction. (C) A collage of ERC exhaustion trials for a single large giant cell held at 0 mV is shown. The primary extinction (▪) and three later secondary extinctions at 500 nm (•, ▴, and ▾) are shown as well as single extinctions at 570 (×) and 430 (♦) nm. In all cases, the flashes promoted a progressive decrease in ERC charge with successive flash number at constant stimulus intensity (500 nm: 4.08 × 108, 430 nm: 3.62 × 108, and 570 nm: 3.38 × 108 photons/μm2). The three extinctions at 500 nm resulted in similar exhaustion curves and Pt estimates. (D) By fitting a single exponential decay model () to data from many experiments (n = 7 cells, 24 extinctions), Pt was determined. The means (±SEM) of Pt are plotted for primary bleaches (1), secondary bleaches (2), and at different wavelengths generated by 70-nm bandwidth filters [500(70), 430(70), and 570(70)] or 30-nm bandwidth filters [500(30)]. Data were collected from five cells held at 0-mV holding potential and two cells held at +30 mV. Means for Pt were not different on the basis of parametric (one-way analysis of variance) and nonparametric (Kruskal-Wallis analysis of variance) tests.
Mentions: Successive flashes at a single wavelength and intensity promoted progressive loss of ERC R2 charge until no further signal was obtained above background current noise. Since the interstimulus intervals between successive flashes were only ∼10 s, pigment regeneration was minimal between stimuli, and regeneration should not contribute to the extinction progression. The spectral sensitivity of the ERC governs not only the efficacy of successive bleaches, but also single bleaches. Fig. 4 A shows the Qi extinction of R2 in response to successive flashes at 570 and then 500 nm in a single giant cell. Cumulative flash intensity delivered is used as the dependent variable. After ERC charge was effectively extinguished into noise by 570-nm flashes, 500-nm flashes of greater effective intensity were immediately delivered. The additional extinguishable ERC charge found with 500-nm flashes indicated residual ground state rhodopsin in the cell after the 570-nm flashes. This resulted because 570-nm flashes are not as effective at eliciting ERC currents as are flashes at 500 nm given the relative absorbance of WT human rhodopsin at 570 vs. 493 nm (peak absorbance) (OD570/OD493 = 0.436) (Wald and Brown 1958). Similar findings occur when bleaching at other wavelengths is followed by flash photolysis at 500 nm. The relative probability of activating rhodopsin, taken as the ratio of absorbance cross sections, is only ∼12% at 570 nm relative to 500 nm (α570/α493 = 0.115), assuming equal photon density at the two wavelengths. At the maximal flash strengths used, the fraction of rhodopsin molecules absorbing at least one photon at 500 vs. 570 nm was estimated to be ∼0.96 and 0.27, respectively. Thus, the flash system does not deliver sufficient photons at 570 nm to compensate for the lower probability of activation. This illustrates that detection of rhodopsin charge motions depends on the unitary charge motion, which should be the same at any wavelength (see univariance below), and the number of activated rhodopsin molecules that mobilize charge and sum into an ensemble ERC current. Even at peak wavelength (≈500 nm), a given flash intensity may not be sufficient to generate ERC currents above noise because the ensemble ERC current lies within the noise band.

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