Time-Resolved Rhodopsin Activation Currents in a Unicellular Expression System

Abstract

The early receptor current (ERC) is the charge redistribution occurring in plasma membrane rhodopsin during light activation of photoreceptors. Both the molecular mechanism of the ERC and its relationship to rhodopsin conformational activation are unknown. To investigate whether the ERC could be a time-resolved assay of rhodopsin structure-function relationships, the distinct sensitivity of modern electrophysiological tools was employed to test for flash-activated ERC signals in cells stably expressing normal human rod opsin after regeneration with 11-cis-retinal. ERCs are similar in waveform and kinetics to those found in photoreceptors. The action spectrum of the major R(2) charge motion is consistent with a rhodopsin photopigment. The R(1) phase is not kinetically resolvable and the R(2) phase, which overlaps metarhodopsin-II formation, has a rapid risetime and complex multiexponential decay. These experiments demonstrate, for the first time, kinetically resolved electrical state transitions during activation of expressed visual pigment in a unicellular environment (single or fused giant cells) containing only 6 x 10(6)-8 x 10(7) molecules of rhodopsin. This method improves measurement sensitivity 7 to 8 orders of magnitude compared to other time-resolved techniques applied to rhodopsin to study the role particular amino acids play in conformational activation and the forces that govern those transitions.

The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin

“…Second, our simulations reveal additional space in hRho (Fig. 3), potentially occupied by an additional water molecule, which influences the vicinity of the Schiff base (47) and thus the UV-visible absorption maximum (48) and the typical infrared absorptions of the dark state. In agreement, the absorption maximum of the SB chimera, in which the bovine 297 TSAV 300 sequence was introduced, was shifted back toward that typical of bRho, whereas the kinetics of Meta II formation remain the same as seen by hRho, suggesting only a local influence of the TSAV region on the retinal Schiff base.…”

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

“…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).…”

Light-induced Reorganization of Phospholipids in Rod Disc Membranes

“…In particular, two explanations arise for the observations with the negatively charged PS. It is known that, with the formation of Meta II, an electric transmembrane potential is generated, the so-called R2 wave of the "early receptor potential" (13). This potential change is superimposed to the transmembrane potential that exists in the dark and is due to the asymmetry of the rhodopsin molecule (see above).…”

“…Electrical effects can be measured in situ, namely, as a component of the "early receptor potential." The most recent development is the "early receptor current" technique, realized in a unicellular expression system (13). Fourier transform infrared spectroscopy showed changes in the molecular environment of lipid after transition of rhodopsin to Meta II (14).…”