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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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Giant cells spontaneously regenerate from intracellular retinal stores. (A) A giant cell regenerated with 11cRet was subjected to ERC exhaustion with 500-nm flashes (4.08 × 108 photons/μm2) and the individual R2 Qi values were integrated from each ERC and plotted versus flash number. Note the similar photolytic exhaustion of the ERC after primary regeneration (▪) and after secondary (spontaneous) recovery (•) during 10 min of dark adaptation. Immediately after the second extinction, 10 mM NH2OH in E-1 (pH 7.0) was perfused to completely replace the bath solution. This extracellular solution was maintained in the bath for the remainder of the experiment. After 5 additional min, 500-nm flashes were delivered again, but many more flashes were needed to slowly decrement the ERC R2 charge into extinction (▴). Two 10 min periods of dark adaptation (*) during exposure to NH2OH permitted some spontaneous regeneration of pigment. (B) A similar but more extensive experiment shows total extinction of the R2 charge in NH2OH on the third bleach. Experimental interventions are drawn above the curve. A 10-min dark adaptation showed essentially no pigment regeneration given minimal ERC charge on the fourth extinction. The chamber was then perfused with E-1 to wash out NH2OH, and then perfused with E-1 containing 25 μM 11cRet in 2% FAF-BSA/0.025% vitamin E. After 10 min of dark adaptation in 11cRet, ERC signals were recovered with a similar level of initial charge (≈400 fC) and similar photosensitivity to bleaching when compared with the first two flash series.
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Figure 2: Giant cells spontaneously regenerate from intracellular retinal stores. (A) A giant cell regenerated with 11cRet was subjected to ERC exhaustion with 500-nm flashes (4.08 × 108 photons/μm2) and the individual R2 Qi values were integrated from each ERC and plotted versus flash number. Note the similar photolytic exhaustion of the ERC after primary regeneration (▪) and after secondary (spontaneous) recovery (•) during 10 min of dark adaptation. Immediately after the second extinction, 10 mM NH2OH in E-1 (pH 7.0) was perfused to completely replace the bath solution. This extracellular solution was maintained in the bath for the remainder of the experiment. After 5 additional min, 500-nm flashes were delivered again, but many more flashes were needed to slowly decrement the ERC R2 charge into extinction (▴). Two 10 min periods of dark adaptation (*) during exposure to NH2OH permitted some spontaneous regeneration of pigment. (B) A similar but more extensive experiment shows total extinction of the R2 charge in NH2OH on the third bleach. Experimental interventions are drawn above the curve. A 10-min dark adaptation showed essentially no pigment regeneration given minimal ERC charge on the fourth extinction. The chamber was then perfused with E-1 to wash out NH2OH, and then perfused with E-1 containing 25 μM 11cRet in 2% FAF-BSA/0.025% vitamin E. After 10 min of dark adaptation in 11cRet, ERC signals were recovered with a similar level of initial charge (≈400 fC) and similar photosensitivity to bleaching when compared with the first two flash series.

Mentions: The source of chromophore that promoted spontaneous ERC recovery was investigated. Single fused giant cells regenerated with 11cRet were subjected to primary extinction by successive flashes at 500 nm in normal bath solution (E-1 without chromophore). Dark adaptation promoted spontaneous pigment regeneration and allowed for a secondary extinction of the ERC. Immediately after the second extinction and throughout the next dark adaptation, the chamber volume was replaced with E-1 containing 10 mM hydroxylamine (NH2OH, pH 7.0) and an additional 5 min of dark adaptation preceded the next series of successive 500-nm flashes. ERCs were elicited and had Qi values comparable with those seen during the primary and secondary extinctions without NH2OH. Qi extinctions before and after NH2OH are shown (Fig. 2 A). When NH2OH was present in the bath many more flashes (in this cell 23 flashes) were required to completely extinguish the ERC R2 charge in comparison to three to five flashes during the primary and secondary extinctions. This suggested that 10 mM NH2OH decreased photosensitivity and this effect will be examined fully in a subsequent study. Two dark adaptations (10 min each) in 10 mM NH2OH were followed by some but not full ERC signal recovery, which was then subsequently extinguished by additional flashes. ERC signals continued to be recordable on the time scale of tens of minutes in the presence of constant 10 mM extracellular NH2OH, a concentration far greater than the initial loading of chromophore (25 μM). Fig. 2 B shows the exhaustion course of Qi vs. flash number for another giant cell before and after introduction of 10 mM NH2OH into the bath solution. In NH2OH total R2 charge decreased with successive extinctions and dark regenerations until no significant signals remained. Bath solution was then replaced with fresh E-1 containing 25 μM 11cRet in 2% FAF-BSA (without NH2OH). Strong ERC R2 signals were regenerated comparable in total charge with that seen during the primary extinction. These experiments demonstrate strong evidence that the source of 11cRet during spontaneous dark regeneration is internal to the cell that is recorded.


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

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

Giant cells spontaneously regenerate from intracellular retinal stores. (A) A giant cell regenerated with 11cRet was subjected to ERC exhaustion with 500-nm flashes (4.08 × 108 photons/μm2) and the individual R2 Qi values were integrated from each ERC and plotted versus flash number. Note the similar photolytic exhaustion of the ERC after primary regeneration (▪) and after secondary (spontaneous) recovery (•) during 10 min of dark adaptation. Immediately after the second extinction, 10 mM NH2OH in E-1 (pH 7.0) was perfused to completely replace the bath solution. This extracellular solution was maintained in the bath for the remainder of the experiment. After 5 additional min, 500-nm flashes were delivered again, but many more flashes were needed to slowly decrement the ERC R2 charge into extinction (▴). Two 10 min periods of dark adaptation (*) during exposure to NH2OH permitted some spontaneous regeneration of pigment. (B) A similar but more extensive experiment shows total extinction of the R2 charge in NH2OH on the third bleach. Experimental interventions are drawn above the curve. A 10-min dark adaptation showed essentially no pigment regeneration given minimal ERC charge on the fourth extinction. The chamber was then perfused with E-1 to wash out NH2OH, and then perfused with E-1 containing 25 μM 11cRet in 2% FAF-BSA/0.025% vitamin E. After 10 min of dark adaptation in 11cRet, ERC signals were recovered with a similar level of initial charge (≈400 fC) and similar photosensitivity to bleaching when compared with the first two flash series.
© Copyright Policy
Related In: Results  -  Collection

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Figure 2: Giant cells spontaneously regenerate from intracellular retinal stores. (A) A giant cell regenerated with 11cRet was subjected to ERC exhaustion with 500-nm flashes (4.08 × 108 photons/μm2) and the individual R2 Qi values were integrated from each ERC and plotted versus flash number. Note the similar photolytic exhaustion of the ERC after primary regeneration (▪) and after secondary (spontaneous) recovery (•) during 10 min of dark adaptation. Immediately after the second extinction, 10 mM NH2OH in E-1 (pH 7.0) was perfused to completely replace the bath solution. This extracellular solution was maintained in the bath for the remainder of the experiment. After 5 additional min, 500-nm flashes were delivered again, but many more flashes were needed to slowly decrement the ERC R2 charge into extinction (▴). Two 10 min periods of dark adaptation (*) during exposure to NH2OH permitted some spontaneous regeneration of pigment. (B) A similar but more extensive experiment shows total extinction of the R2 charge in NH2OH on the third bleach. Experimental interventions are drawn above the curve. A 10-min dark adaptation showed essentially no pigment regeneration given minimal ERC charge on the fourth extinction. The chamber was then perfused with E-1 to wash out NH2OH, and then perfused with E-1 containing 25 μM 11cRet in 2% FAF-BSA/0.025% vitamin E. After 10 min of dark adaptation in 11cRet, ERC signals were recovered with a similar level of initial charge (≈400 fC) and similar photosensitivity to bleaching when compared with the first two flash series.
Mentions: The source of chromophore that promoted spontaneous ERC recovery was investigated. Single fused giant cells regenerated with 11cRet were subjected to primary extinction by successive flashes at 500 nm in normal bath solution (E-1 without chromophore). Dark adaptation promoted spontaneous pigment regeneration and allowed for a secondary extinction of the ERC. Immediately after the second extinction and throughout the next dark adaptation, the chamber volume was replaced with E-1 containing 10 mM hydroxylamine (NH2OH, pH 7.0) and an additional 5 min of dark adaptation preceded the next series of successive 500-nm flashes. ERCs were elicited and had Qi values comparable with those seen during the primary and secondary extinctions without NH2OH. Qi extinctions before and after NH2OH are shown (Fig. 2 A). When NH2OH was present in the bath many more flashes (in this cell 23 flashes) were required to completely extinguish the ERC R2 charge in comparison to three to five flashes during the primary and secondary extinctions. This suggested that 10 mM NH2OH decreased photosensitivity and this effect will be examined fully in a subsequent study. Two dark adaptations (10 min each) in 10 mM NH2OH were followed by some but not full ERC signal recovery, which was then subsequently extinguished by additional flashes. ERC signals continued to be recordable on the time scale of tens of minutes in the presence of constant 10 mM extracellular NH2OH, a concentration far greater than the initial loading of chromophore (25 μM). Fig. 2 B shows the exhaustion course of Qi vs. flash number for another giant cell before and after introduction of 10 mM NH2OH into the bath solution. In NH2OH total R2 charge decreased with successive extinctions and dark regenerations until no significant signals remained. Bath solution was then replaced with fresh E-1 containing 25 μM 11cRet in 2% FAF-BSA (without NH2OH). Strong ERC R2 signals were regenerated comparable in total charge with that seen during the primary extinction. These experiments demonstrate strong evidence that the source of 11cRet during spontaneous dark regeneration is internal to the cell that is recorded.

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