Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Full Text Full Text (PDF, 344K) PPT slides of all figures Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JGP Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Shukla, P. Articles by Sullivan, J. M. Search for Related Content PubMed PubMed Citation Articles by Shukla, P. Articles by Sullivan, J. M. Social Bookmarking                            What's this?

Original Article

^a From the Department of Ophthalmology, State University of New York, Health Science Center at Syracuse, Syracuse, New York 13210
^b From the Department of Biochemistry, State University of New York, Health Science Center at Syracuse, Syracuse, New York 13210
Assistant Professor of Ophthalmology and Biochemistry, SUNY Health Science Center, Dept. of Biochemistry Weiskotten Hall, 4255, 750 East Adams St., Syracuse, NY 13210.Fax: 315-464-8750;

sullivaj{at}vax.cs.hscsyr.edu

The early receptor current (ERC) represents molecular charge movement during rhodopsin conformational dynamics. To determine whether this time-resolved assay can probe various aspects of structure–function relationships in rhodopsin, we first measured properties of expressed normal human rhodopsin with ERC recordings. These studies were conducted in single fused giant cells containing on the order of a picogram of regenerated pigment. The action spectrum of the ERC of normal human opsin regenerated with 11-cis-retinal was fit by the human rhodopsin absorbance spectrum. Successive flashes extinguished ERC signals consistent with bleaching of a rhodopsin photopigment with a normal range of photosensitivity. ERC signals followed the univariance principle since millisecond-order relaxation kinetics were independent of the wavelength of the flash stimulus. After signal extinction, dark adaptation without added 11-cis-retinal resulted in spontaneous pigment regeneration from an intracellular store of chromophore remaining from earlier loading. After the ERC was extinguished, 350-nm flashes overlapping metarhodopsin-II absorption promoted immediate recovery of ERC charge motions identified by subsequent 500-nm flashes. Small inverted R₂ signals were seen in response to some 350-nm flashes. These results indicate that the ERC can be photoregenerated from the metarhodopsin-II state. Regeneration with 9-cis-retinal permits recording of ERC signals consistent with flash activation of isorhodopsin. We initiated structure–function studies by measuring ERC signals in cells expressing the D83N and E134Q mutant human rhodopsin pigments. D83N ERCs were simplified in comparison with normal rhodopsin, while E134Q ERCs had only the early phase of charge motion. This study demonstrates that properties of normal rhodopsin can be accurately measured with the ERC assay and that a structure–function investigation of rapid activation processes in analogue and mutant visual pigments is feasible in a live unicellular environment.

Key Words: photoreceptor • gating currents • conformational activation • phototransduction

CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				M. L. Woodruff, J. Lem, and G. L. Fain Early receptor current of wild-type and transducin knockout mice: photosensitivity and light-induced Ca2+ release J. Physiol., June 15, 2004; 557(3): 821 - 828. [Abstract] [Full Text] [PDF]

				R. Herrmann, M. Heck, P. Henklein, P. Henklein, C. Kleuss, K. P. Hofmann, and O. P. Ernst Sequence of Interactions in Receptor-G Protein Coupling J. Biol. Chem., June 4, 2004; 279(23): 24283 - 24290. [Abstract] [Full Text] [PDF]

				L. I. Brueggemann and J. M. Sullivan HEK293S Cells Have Functional Retinoid Processing Machinery J. Gen. Physiol., June 1, 2002; 119(6): 593 - 612. [Abstract] [Full Text] [PDF]

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents