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P. N. Itotia, J. M. Sullivan; Quantal Efficiency of Rhodopsin Activation Measured With the Early Receptor Current. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1277. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To establish the early receptor current (ERC) of rhodopsin activation as a measure of quantal efficiency (QE) in expressed visual pigments.
WT human rod opsin was expressed in HEK293S cells for ERC investigation as previously described (Sullivan and Shukla, 1999, Biophys J. 77:1333-1357). Single fused giant cells were regenerated with 11-cis-retinal (11cRet) to form ground state pigments. HEK293 cells express retinoid processing machinery that regenerates ground state chromophores after bleaching without added 11cRet (Brueggemann and Sullivan, 2002, J. Gen. Physiol. 119:593-612). Whole-cell patch clamp electrophysiology measured ERC R2 currents (msec-order Meta-II formation) under conditions of variable intensity flash photolysis at 500 nm (± 35 nm). Flash intensity was varied with neutral density filters and modulation of flash capacitor charge voltage. Flash duration is 14 µsec (Sullivan, 1998, Rev. Sci. Instr. 69: 527-539). ERC charge motions were acquired with serial, constant intensity flashes until no further knockdown of ERC charge was observed. At each stimulus intensity, single exponential extinction curves were fit to the charge decrement data. The photosensitivity parameter (Pt) was extracted from the fit parameter. Pt is the product of molecular cross section (α) and QE (γ).
Due to the intrinsic retinoid processing machinery, multiple bleach/recovery cycles of ERC data could be acquired from each giant cell. Extinction data was uniform from one cycle to the next when allowing 10 minutes between bleaches for consistent pigment regeneration. ERC charge motions suppressed with serial flash photolysis leaving a small fraction (<0.15) of charge which was not extinguished. High intensity flashes extinguished approximately 0.5 of the remaining charge with each flash. Mean Pt values decreased with increases in flash intensity. At the lowest flash intensities Pt was approximately 1x10-8 µm2 and consistent with the peak absorption coefficient of human rhodopsin, but decreased approximately 1 log at the highest flash intensities. A decrease of Pt with intensity suggests a decrease in γ, α or αγ as photon absorption probability increases.
Photoconversion from bathorhodopsin547nm or lumirhodospin497nm during the 14 µsec flash duration can repopulate the ground state and modulate Pt due to even numbered absorptions. Investigation of the influence on Pt of stimulus bandwidth and duration are ongoing. Extension to retinoid binding pocket mutants (G90D) and expressed human cone pigments are straightforward.
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