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 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.
Sullivan
J M
JM
Department of Ophthalmology, State University of New York, Health Science Center at Syracuse, Syracuse, New York 13210 USA. Sullivaj@vax.cs.hscsyr.edu
Shukla
P
P
D002459:Cell Fusion; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D004594:Electrophysiology / Q000379:methods; D006801:Humans; D007700:Kinetics; D008027:Light; D008564:Membrane Potentials / Q000502:physiology; D008954:Models, Biological; D010782:Photolysis; D011994:Recombinant Proteins / Q000096:biosynthesis; D017948:Retinal Rod Photoreceptor Cells / Q000502:physiology*; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry / Q000502:physiology* / Q000528:radiation effects; D017299:Rod Opsins / Q000235:genetics / Q000502:physiology; D013997:Time Factors; D014162:Transfection
D011994:Recombinant Proteins; D017299:Rod Opsins; D012243:Rhodopsin; D012172:Retinaldehyde