An Additional Retinoid Binding Site in Rhodopsin

Abstract

Recently we discovered that some dark-adapted salamander rods and cones generated an electrical response to the truncated retinal analogue, β-ionone. This finding was tempered by observations that exposure to β-ionone led to pigment bleaching, and that very high concentrations of β-ionone inhibited rod channels in patch experiments. Therefore we examined whether β-ionone could activate the visual pigments by attacking the chromophore-binding pocket or through an interaction at an alternate binding site. Microspectrophotometry showed an accumulation of β-ionone in green-sensitive rod outer segments that increased linearly with bath concentration indicating that β-ionone most likely partitioned into disk membranes. β-Ionone also went into blue-sensitive rod outer segments, however, uptake was higher than in green-sensitive rods at all bath concentrations tested, suggesting that β-ionone bound to at least one site on rhodopsin in addition to partitioning. X-ray diffraction of green-sensitive rod rhodopsin crystallized in the presence of millimolar concentrations of β-ionone revealed a binding site located near the extracellular/intradiskal side of rhodopsin. β-Ionone may have low efficacy and low binding affinity because rhodopsin with ligand bound retained the inactive state conformation. β-Ionone is not a native retinoid, so we also examined the effects of retinol. Dark-adapted green-sensitive rods exposed to retinol lost sensitivity to flashes due to direct rhodopsin activation. In addition, rods exhibited a relative increase in sensitivity to shorter wavelengths, consistent with the ability of retinol to act as an antenna chromophore. Similar effects were seen for a blue-sensitive rod. These results support the presence in opsins of a retinoid-binding site(s) in addition to the chromophore-binding pocket, and suggest that this alternate site(s) mediates a number of distinct ligand-specific effects.