Abstract
Abstract: :
Purpose: To examine fast conformational activation in G90D rhodopsin. This is one of a few human rod opsin mutations (G90D, T94I, A292E) that occur in the immediate environment of the protonated Schiff base (PSB) of the chromophore to promote a state of rod desensitization. Methods: The early receptor current (ERC) assay is used to investigate the electrical events associated with late conformational activation of human wild type (WT), G90D, and E113Q rod rhodopsins that are heterologously expressed in HEK293 cells. Results: In WT rhodopsin successive flashes progressively deplete and eventually extinguish outward msec-order R2 charge motion associated with metarhodopsin-II formation. G90D is unique in that a photostationary state (PSS) is established upon successive 500 nm flashes. Upon the first flash G90D generates a brief outward R2 charge motion. The second and third flashes lead to progressive and complete inversion of R2 charge motion to populate a PSS that resists charge extinction (bleaching) upon further stimuli. The inward R2 of the G90D PSS is similar to the R2 signal of the PSB counterion mutant, E113Q (or WT at low extracellular pH (pHo)). While WT R2 is sensitive to pHo with two titrations (outward: pKa = 6.8; inward pKa ~ 4.5), the G90D PSS pHo sensitivity has an anomalous purely inward titration. The PSS of G90D R2 senses intracellular pH (pHi) (pKa = 6.8) but the dynamic range is compressed relative to WT. Unlike WT, where pHi sensitive proton uptake dominates late R2, there is little effect of pHi on R2 relaxation rate in the G90D PSS. Chemical or structural changes measured as WT R2 sense the transmembrane electrical field but this sensitivity is lost in the G90D PSS. All- trans-retinal regeneration simulates the inward charge motion of the PSS established from an 11-cis-retinal regenerated G90D pigment, but the pigment bleaches. The action spectrum of the PSS in G90D regenerated with 11-cis-retinal (peak 437 nm) is similar to the pigment formed with all-trans-retinal (peak 450 nm). This suggests that the PSS in G90D results from an all-trans-retinylidene chromophore with a PSB. Conclusions: We hypothesize that the establishment of the G90D PSS in membranes relates to both chromophore isomerization, a shift in the PSB counterion from E113 to G90D and conformational rearrangements to form a pigment that resists bleaching. The persistence of the G90D PSS may result because the PSB environment is protected from hydrolysis and/or has a high quantal efficiency of reverse photoconversion from intermediate states on the forward activation path. The long-lived PSS may relate to elevated rod psychophysical thresholds in G90D patients following light exposure.
Keywords: retinal degenerations: hereditary • opsins • dark/light adaptation