May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Properties of Rhodopsin Regeneration in HEK293S Cells
Author Affiliations & Notes
  • N. Bath
    Ophthalmology, SUNY at Buffalo, Buffalo, New York
  • P. N. Itotia
    Ophthalmology, SUNY at Buffalo, Buffalo, New York
  • J. M. Sullivan
    Ophthalmology, SUNY at Buffalo, Buffalo, New York
  • Footnotes
    Commercial Relationships N. Bath, None; P.N. Itotia, None; J.M. Sullivan, None.
  • Footnotes
    Support NIH Grant EY11384, Research to Prevent Blindness, Oishei Foundation
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 3248. doi:
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      N. Bath, P. N. Itotia, J. M. Sullivan; Properties of Rhodopsin Regeneration in HEK293S Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3248.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: To determine the extent to which inhibition of RPE65 and plasma membrane voltage influence visual pigment regeneration in HEK293S cells stably expressing human rod opsin (Rho). Inhibitors of RPE65 such as 13-cis-retinoic acid (13cRA) and 2,2’-dipyridyl (2,2Di) allow testing of the role of this protein, known to be expressed in abundance in HEK293S cells (Ma et al., 1999; Chen et al., 2003), in the recurrent visual pigment regeneration or dark adaptation following serial bleaching by flash photolysis (Brueggemann and Sullivan, 2002).

Methods:: HEK293S cells expressing wild type (WT) Rho were regenerated with 11-cis-retinal or all-trans-retinal in the dark. Early receptor currents (ERCs) or Rho gating currents were recorded under established conditions (Shukla and Sullivan, 1999; Brueggemann and Sullivan, 2002). ERCs were elicited by intense flash photolysis at 500 nm. ERCs were extinguished by serial flashes and then dark adaptation without added retinoid, promoted recovery of WT ERC signal. After recording ERCs in standard intracellular (I12) and extracellular (E1) buffers, E1 containing bovine serum albumin (BSA) was perfused, and then 13cRA in different concentrations was perfused mixed in E1 with BSA. ERCs were recorded over time during the serial solution changes and the impact of the different solutions on WT ERC charge motion determined and statistically analyzed. The impact of the iron chelator 2,2’-dipyridyl on recurrent Rho regeneration was determined by including the agent in the recording pipette for intracellular dialysis.

Results:: 13cRA (Isotretinoin) exerts a time dependent and concentration dependent block of recurrent WT Rho regeneration in HEK293S cells. 13cRA begins to inhibit ERC charge during its perfusion and continues to promote further charge reduction after its washout. The inhibition occurs more rapidly when Rho turnover is increased by more frequent bleaching epochs. 2,2Di exerts a similar effect to 13cRA upon perfusion into the cytoplasm of the HEK293S cell. While retinals have substantial dipole moments, varying constant plasma membrane voltage has little, if any, effect on the amount of ERC charge regenerated.

Conclusions:: Known inhibitors of RPE65, 13cRA and 2,2Di, exert strong inhibition on Rho regeneration in HEK293S cells. This data suggests that the retinoid isomerase RPE65 plays a prominent role in the recovery of 11-cis-retinaldehyde needed to regenerated ground state pigment in WT-HEK293S cells. Isotretinoin has been suggested as a drug to slow dry age related macular degeneration. The HEK293S regeneration system may provide a useful model system to study the impact of this drug on retinoid metabolism.

Keywords: retinoids/retinoid binding proteins • opsins • enzymes/enzyme inhibitors 
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