Time-resolved rhodopsin activation currents in a unicellular expression system

The early receptor current (ERC) is the charge redistribution occurring in plasma membrane rhodopsin during light activation of photoreceptors, Both t he molecular mechanism of the ERC and its relationship to rhodopsin conform ational activation are unknown. To investigate whether the ERC could be a t ime-resolved assay of rhodopsin structure-function relationships, the disti nct sensitivity of modern electrophysiological tools was employed to test f or 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 m ajor 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 meta rhodopsin-11 formation, has a rapid risetime and complex multiexponential d ecay. These experiments demonstrate, for the first time, kinetically resolv ed electrical state transitions during activation of expressed visual pigme nt in a unicellular environment (single or fused giant cells) containing on ly 6 x 10(6)-8 x 10(7) molecules of rhodopsin. This method improves measure ment sensitivity 7 to 8 orders of magnitude compared to other time-resolved techniques applied to rhodopsin to study the role particular amino acids p lay in conformational activation and the forces that govern those transitio ns.