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HEK293S cells have functional retinoid processing machinery.

Brueggemann LI, Sullivan JM - J. Gen. Physiol. (2002)

Bottom Line: Rhodopsin activation is measured by the early receptor current (ERC), a conformation-associated charge motion, in human embryonic kidney cells (HEK293S) expressing opsins.This indicates conversion of loaded Vitamin A or all-trans-retinal into cis-retinaldehyde that regenerated ground-state pigment. 4-butylaniline, an inhibitor of the mammalian retinoid cycle, reversibly suppressed recovery of the outward R(2) component from Vitamin A and 11-cis-retinal-loaded cells.These physiological findings are evidence for the presence of intrinsic retinoid processing machinery in WT-HEK293S cells similar to what occurs in the mammalian eye.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Institute for Human Performance, State University of New York, Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.

ABSTRACT
Rhodopsin activation is measured by the early receptor current (ERC), a conformation-associated charge motion, in human embryonic kidney cells (HEK293S) expressing opsins. After rhodopsin bleaching in cells loaded with 11-cis-retinal, ERC signals recover in minutes and recurrently over a period of hours by simple dark adaptation, with no added chromophore. The purpose of this study is to investigate the source of ERC signal recovery in these cells. Giant HEK293S cells expressing normal wild-type (WT)-human rod opsin (HEK293S) were regenerated by solubilized 11-cis-retinal, all-trans-retinal, or Vitamin A in darkness. ERCs were elicited by flash photolysis and measured by whole-cell recording. Visible flashes initially elicit bimodal (R(1), R(2)) ERC signals in WT-HEK293S cells loaded with 11-cis-retinal for 40 min or overnight. In contrast, cells regenerated for 40 min with all-trans-retinal or Vitamin A had negative ERCs (R(1)-like) or none at all. After these were placed in the dark overnight, ERCs with outward R(2) signals were recorded the following day. This indicates conversion of loaded Vitamin A or all-trans-retinal into cis-retinaldehyde that regenerated ground-state pigment. 4-butylaniline, an inhibitor of the mammalian retinoid cycle, reversibly suppressed recovery of the outward R(2) component from Vitamin A and 11-cis-retinal-loaded cells. These physiological findings are evidence for the presence of intrinsic retinoid processing machinery in WT-HEK293S cells similar to what occurs in the mammalian eye.

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ERC signals from WT-HEK293S cells regenerated with different cis-retinaldehydes. Fused WT-HEK293S giant cells were loaded with 25 μM 11-cis-retinal or 9-cis-retinal or 50 μM 13-cis-retinal complexed to FAF-BSA. ERC signals on the first 500-nm flash during the primary bleaching extinction (A, C, and E) and secondary bleaching extinctions (B, D, and F) are shown for 11-cis-retinal– (A and B), 9-cis-retinal– (C and D), and 13-cis-retinal– (E and F) loaded representative cells. Membrane capacitances of the cells are indicated. The arrow indicates the timing of the flash stimulus. Responses from each single cell are representative of larger populations of cells regenerated with 11-cis-retinal (n = 54), 9-cis-retinal (n = 5), and 13-cis-retinal (n = 4).
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fig2: ERC signals from WT-HEK293S cells regenerated with different cis-retinaldehydes. Fused WT-HEK293S giant cells were loaded with 25 μM 11-cis-retinal or 9-cis-retinal or 50 μM 13-cis-retinal complexed to FAF-BSA. ERC signals on the first 500-nm flash during the primary bleaching extinction (A, C, and E) and secondary bleaching extinctions (B, D, and F) are shown for 11-cis-retinal– (A and B), 9-cis-retinal– (C and D), and 13-cis-retinal– (E and F) loaded representative cells. Membrane capacitances of the cells are indicated. The arrow indicates the timing of the flash stimulus. Responses from each single cell are representative of larger populations of cells regenerated with 11-cis-retinal (n = 54), 9-cis-retinal (n = 5), and 13-cis-retinal (n = 4).

Mentions: Retinoids are required to obtain ERC signals. Once retinoids are loaded and removed giant WT-HEK293S cells were not subsequently exposed to retinoids, unless explicitly stipulated. Visual pigment recovery after bleaching must occur from available retinoid substrates initially loaded into cells. WT-HEK293S cells regenerated with naturally occurring cis-retinaldehydes 11-cis-retinal (Fig. 2 A), 9-cis-retinal (Fig. 2 C), or 13-cis-retinal (Fig. 2 E) all yield whole-cell ERCs having both bimodal inward (R1) and outward (R2) currents on the initial 500-nm flash photolysis series. The R2 charge motion associates with formation of the Meta-II conformational states during forward bleaching of rhodopsin (Spalink and Stieve, 1980; Makino et al., 1991; Sullivan and Shukla, 1999; Shukla and Sullivan, 1999; unpublished data). All signals extinguish by serial flash photolysis. WT-HEK293S cells loaded with either 11-cis- or 9-cis-retinal regenerate pigments yielding large, inward R1 and outward R2 charge motions upon initial flash photolysis similar to ERCs of rod and cone photoreceptors (Makino et al., 1991; Sullivan et al., 2000). These chromophores regenerate stable ground state pigments, rhodopsin and isorhodopsin, respectively, which bleach by an identical series of conformational intermediates that promote charge motions (Kliger et al., 1984). In contrast, 13-cis-retinal, the chromophore of dark-adapted bacteriorhodopsin, regenerates a small but definitive R2 signal upon the initial bleach series despite a large R1 signal.


HEK293S cells have functional retinoid processing machinery.

Brueggemann LI, Sullivan JM - J. Gen. Physiol. (2002)

ERC signals from WT-HEK293S cells regenerated with different cis-retinaldehydes. Fused WT-HEK293S giant cells were loaded with 25 μM 11-cis-retinal or 9-cis-retinal or 50 μM 13-cis-retinal complexed to FAF-BSA. ERC signals on the first 500-nm flash during the primary bleaching extinction (A, C, and E) and secondary bleaching extinctions (B, D, and F) are shown for 11-cis-retinal– (A and B), 9-cis-retinal– (C and D), and 13-cis-retinal– (E and F) loaded representative cells. Membrane capacitances of the cells are indicated. The arrow indicates the timing of the flash stimulus. Responses from each single cell are representative of larger populations of cells regenerated with 11-cis-retinal (n = 54), 9-cis-retinal (n = 5), and 13-cis-retinal (n = 4).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233870&req=5

fig2: ERC signals from WT-HEK293S cells regenerated with different cis-retinaldehydes. Fused WT-HEK293S giant cells were loaded with 25 μM 11-cis-retinal or 9-cis-retinal or 50 μM 13-cis-retinal complexed to FAF-BSA. ERC signals on the first 500-nm flash during the primary bleaching extinction (A, C, and E) and secondary bleaching extinctions (B, D, and F) are shown for 11-cis-retinal– (A and B), 9-cis-retinal– (C and D), and 13-cis-retinal– (E and F) loaded representative cells. Membrane capacitances of the cells are indicated. The arrow indicates the timing of the flash stimulus. Responses from each single cell are representative of larger populations of cells regenerated with 11-cis-retinal (n = 54), 9-cis-retinal (n = 5), and 13-cis-retinal (n = 4).
Mentions: Retinoids are required to obtain ERC signals. Once retinoids are loaded and removed giant WT-HEK293S cells were not subsequently exposed to retinoids, unless explicitly stipulated. Visual pigment recovery after bleaching must occur from available retinoid substrates initially loaded into cells. WT-HEK293S cells regenerated with naturally occurring cis-retinaldehydes 11-cis-retinal (Fig. 2 A), 9-cis-retinal (Fig. 2 C), or 13-cis-retinal (Fig. 2 E) all yield whole-cell ERCs having both bimodal inward (R1) and outward (R2) currents on the initial 500-nm flash photolysis series. The R2 charge motion associates with formation of the Meta-II conformational states during forward bleaching of rhodopsin (Spalink and Stieve, 1980; Makino et al., 1991; Sullivan and Shukla, 1999; Shukla and Sullivan, 1999; unpublished data). All signals extinguish by serial flash photolysis. WT-HEK293S cells loaded with either 11-cis- or 9-cis-retinal regenerate pigments yielding large, inward R1 and outward R2 charge motions upon initial flash photolysis similar to ERCs of rod and cone photoreceptors (Makino et al., 1991; Sullivan et al., 2000). These chromophores regenerate stable ground state pigments, rhodopsin and isorhodopsin, respectively, which bleach by an identical series of conformational intermediates that promote charge motions (Kliger et al., 1984). In contrast, 13-cis-retinal, the chromophore of dark-adapted bacteriorhodopsin, regenerates a small but definitive R2 signal upon the initial bleach series despite a large R1 signal.

Bottom Line: Rhodopsin activation is measured by the early receptor current (ERC), a conformation-associated charge motion, in human embryonic kidney cells (HEK293S) expressing opsins.This indicates conversion of loaded Vitamin A or all-trans-retinal into cis-retinaldehyde that regenerated ground-state pigment. 4-butylaniline, an inhibitor of the mammalian retinoid cycle, reversibly suppressed recovery of the outward R(2) component from Vitamin A and 11-cis-retinal-loaded cells.These physiological findings are evidence for the presence of intrinsic retinoid processing machinery in WT-HEK293S cells similar to what occurs in the mammalian eye.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Institute for Human Performance, State University of New York, Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.

ABSTRACT
Rhodopsin activation is measured by the early receptor current (ERC), a conformation-associated charge motion, in human embryonic kidney cells (HEK293S) expressing opsins. After rhodopsin bleaching in cells loaded with 11-cis-retinal, ERC signals recover in minutes and recurrently over a period of hours by simple dark adaptation, with no added chromophore. The purpose of this study is to investigate the source of ERC signal recovery in these cells. Giant HEK293S cells expressing normal wild-type (WT)-human rod opsin (HEK293S) were regenerated by solubilized 11-cis-retinal, all-trans-retinal, or Vitamin A in darkness. ERCs were elicited by flash photolysis and measured by whole-cell recording. Visible flashes initially elicit bimodal (R(1), R(2)) ERC signals in WT-HEK293S cells loaded with 11-cis-retinal for 40 min or overnight. In contrast, cells regenerated for 40 min with all-trans-retinal or Vitamin A had negative ERCs (R(1)-like) or none at all. After these were placed in the dark overnight, ERCs with outward R(2) signals were recorded the following day. This indicates conversion of loaded Vitamin A or all-trans-retinal into cis-retinaldehyde that regenerated ground-state pigment. 4-butylaniline, an inhibitor of the mammalian retinoid cycle, reversibly suppressed recovery of the outward R(2) component from Vitamin A and 11-cis-retinal-loaded cells. These physiological findings are evidence for the presence of intrinsic retinoid processing machinery in WT-HEK293S cells similar to what occurs in the mammalian eye.

Show MeSH
Related in: MedlinePlus