Normal and Mutant Rhodopsin Activation Measured with the Early Receptor Current in a Unicellular Expression System

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 R2 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.

Development of lead hammerhead ribozyme candidates against human rod opsin mRNA for retinal degeneration therapy

“…In this stable cell line the opsin cDNA is expressed from a CMV promoter/enhancer. The steady state value of 5 × 10 6 opsin molecules/cell is consistent with our prior measures by spectrophotometry and electrophysiology in this same exact cell line (Sullivan and Shukla, 1999; Shukla and Sullivan, 1999; Sullivan and Satchwell, 2000). In a transient transfection experiment, as used in the current set of experiments, there is at least 20 μg of opsin synthesized in a 48 hr period post transfection.…”

“…Nevertheless, our measures are consistent with a direct linear proportionality between the numbers of opsins present and the Cy5 volume counts. The minimum number of Cy5-labeled antibody molecules detectable was 2.86 × 10 8 , which corresponds to detection levels of RHO in approximately 50 cells of a stable expressing cell line (Sullivan and Shukla, 1999; Shukla and Sullivan, 1999). From the slope of the fitted line we determined that the sensitivity of detection is 1.08 × 10 −5 fluorescent counts/Cy5 molecule.…”

“…The mean raw Cy5 volume counts (not normalized to controls) obtained from opsin measures on western blots used to obtain Figs 8B and 8C were all within the linear range of direct Cy5 labeled antibody measures on the Storm 860 platform. In addition, given the expected level of opsin expression in transfected HEK293S cells (~5 × 10 6 opsins/cell; Sullivan and Satchwell, 2000; Sullivan and Shukla, 1999; Shukla and Sullivan, 1999), and assuming a 1:1:1 relationship between opsin molecules on the membrane, 1D4 molecules bound to opsin, and Cy5 secondary molecules bound to 1D4, the measured slope of the Cy5 secondary antibody measure curve (6.52 volume counts/attomole Cy5 or 1.08 × 10 −5 volume counts/molecule) we estimate a fluorescent output for RHO that is on the experimentally-determined linear dynamic range of Cy5 antibody measure. To minimize dominant sources of variation in western analyses, all experiments were conducted by the same individual (HA), and a single stable fluorescent secondary antibody staining solution preparation was used throughout the study (Koller and Watzig, 2005; Watzig, 2005).…”

Sequence of Interactions in Receptor-G Protein Coupling

“…Such dimers do not exist in the single photon working range (36) of the rod photoreceptor cell. Furthermore, it remains to be studied how the sequence of catalytic steps in R*-G protein coupling identified here relate to different conformations of the activated receptor, which are separated by protonation changes in rhodopsin (2,(37)(38)(39)(40)(41). The G protein may use different receptor conformations, which can bind either CT␣ or CT␥-far, to allosterically control the affinity of the two binding sites on the receptor for efficient nucleotide exchange catalysis.…”

Early receptor current of wild‐type and transducin knockout mice: photosensitivity and light‐induced Ca2+ release

“…The change in current that produces the ERP, called the early receptor current or ERC, has also been recorded from lower vertebrates with voltage‐clamp recording (Hestrin & Korenbrot, 1990; Makino et al 1991; Makino & Dodd, 1996). Although the ERC of human rhodopsin has been studied in an expression system (Shukla & Sullivan, 1999; Brueggemann & Sullivan, 2001), no attempt has been made to record the ERC from an intact photoreceptor of a mammal. Since mouse has become the most useful vertebrate for studying physiological effects of mutations in rhodopsin and other transduction proteins, we have attempted to measure the ERC of mouse rods, hoping that recordings of a mammalian ERC might provide a further tool for the analysis of models of retinal degeneration and perhaps also provide a method for relating changes in pigment in a mouse model to changes in the ERP waveform or amplitude recorded from patients in a clinical setting.…”