Rhodopsin early receptor currents following flash photolysis of single cells expressing human rhodopsin

Purpose. The early receptor current (ERC) reflects the charge redistribution during activation of rhodopsin. To investigate whether the ERC could be used as a time-resolved assay of structure-function relationships in flash activated rhodopsin, we exploited the sensitivity of the whole cell patch clamp technique to test for ERCs in single cells heterologously expressing human wild type opsin that was regenerated with 11-cis-retinal (11cR). Methods. HEK293 cells constitutively expressing high plasmamembrane levels of opsin were used for electrophysiological studies. Transient whole-cell currents were measured following stimulation by a microbeam flash photolysis unit. Results. Single opsin-expressing HEK293 cells regenerated with 11cR demonstrate ERCs following microsecond flash photolysis. The amount of regenerable opsin in this line implies that ERC signals result from activation of subpicogram quantities of pigment and demonstrates the high sensitivity of the method in acquiring time-resolved data. Expression cell ERCs are similar in temporal waveform (R2 phase) to those found in intact photoreceptors. Over successive flashes the ERC signals decrement until extinction, consistent with bleaching of regenerated photopigment. Flash-extinguished ERCs can be regenerated by local perfusion of 11cR indicating that the opsin can be photocycled in a given cell. Conclusions. ERCs can be acquired from single HEK293 cells expressing human opsin to high plasmamembrane levels. Expression ERCs are a new time-resolved assay of rhodopsin conformational dynamics during activation. Site-specific opsin mutants can also be expressed to high levels in HEK293 cells permitting study of the role particular amino acids play in charge redistribution during activation. Since over 90 rod opsin mutations cause autosomal dominant retinitis pigmentosa, this assay will be used to probe the molecular dysfunction of mutant human pigments that can regenerate with 11cR.