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.
Highlights
Rhodopsin is the visual pigment of the rod photoreceptor and catalyzes the activation of the G-protein, transducin
The early receptor current (ERC) originated from plasma membrane opsin that was regenerated with chromophore and the amount of charge motion was proportional to the size of the fused giant cell, consistent with the quantity of opsin expressed in the plasma membrane
ERCs of two mutant pigments D83N and E134Q demonstrate qualitative and quantitative differences with respect to WT ERCs. These results strongly suggest that the expression ERC approach could be productively expanded to investigate a broad range of rhodopsin activation properties of analogue visual pigments and mutant visual pigments, embracing a structure–function approach applied to both the chromophore and remote environments of rhodopsin
Summary
Rhodopsin is the visual pigment of the rod photoreceptor and catalyzes the activation of the G-protein, transducin. At least two critical and sequential proton exchange mechanisms occur during Meta-II formation: the net transfer of the Schiff base proton to the counterion at E113Ϫ and the uptake of two protons into the cytoplasmic membrane surface (Parkes and Liebman, 1984; Arnis et al, 1994; Jager et al, 1994). These charge motions are reflected in transitions between at least two respective Meta-II states (Meta-IIa and Meta-IIb) that share the same absorption (max 380 nm).
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