Activation of Transducin by a Xenopus Short Wavelength Visual Pigment

Abstract

Phototransduction in cones differs significantly from that in rods in sensitivity, kinetics, and recovery following exposure to light. The contribution that the visual pigment makes in determining the cone response was investigated biochemically by expressing a Xenopus violet cone opsin (VCOP) cDNA in COS1 cells and assaying the light-dependent activation of transducin. Light-exposed VCOP stimulated [35 S]guanosine 5-(␥-thio)triphosphate nucleotide exchange on bovine rod transducin in a time-dependent manner with a half-time for activation of 0.75 min, similar to that of bovine rhodopsin. In exhaustive binding assays, VCOP and rhodopsin activity showed similar concentration dependence with half-maximal activation occurring at 0.02 mol of pigment/mol of transducin. Although VCOP was able to activate as many as 12 transducins per photoisomerization, rhodopsin catalyzed significantly more. When assays were performed with > 420 nm illumination, VCOP exhibited rapid regeneration and high affinity for the photoregenerated 11-cis-retinal. Recycling of the chromophore and reactivation of the pigment resulted in multiple activations of transducin, whereas a maximum of 1 transducin per VCOP was activated under brief illumination. The decay of the active species formed following photobleaching was complete in <5 min, ϳ10-fold faster than that of rhodopsin. In vitro, VCOP activated rod transducin with kinetics and affinity similar to those of rhodopsin, but the active conformation decayed more rapidly and the apoprotein regenerated more efficiently with VCOP than with rhodopsin. These properties of the violet pigment may account for much of the difference in response kinetics between rods and cones.Photopic vision is mediated by specialized photoreceptor cone cells that function at high levels of illumination, respond to rapid changes in light, and permit color discrimination (1, 2). Each cone cell expresses a cone opsin. The cone opsins are members of a larger family of visual pigments (3-5) and share significant amino acid sequence homology with the rod pigment rhodopsin (6). Among the cone pigments, the short wavelength pigments (Group S, with wavelengths of peak absorbance ( max ) 1 ϳ 415-440 nm) permit vision in the violet/blue region of the spectrum and are represented by the mammalian blue, chicken violet, and Xenopus violet pigments.2 Although cone cells expressing Group S pigments are in the minority in the vertebrate retina, they are an integral part of vision. For example, in blue monochromats, i.e. humans with red-green pigment mutations, the blue opsin is the sole mediator of photopic vision (7,8).The phototransduction mechanisms have been extensively investigated in rods (for reviews see Refs. 2 and 9). Following absorption of light and isomerization of 11-cis-retinal to alltrans-retinal, rhodopsin undergoes a series of conformational changes that eventually leads to a transient state, coincident with metarhodopsin(II) (MetaII), that activates the second messenger cascade (9). MetaII interacts with the heterot...

Low amplification and fast visual pigment phosphorylation as mechanisms characterizing cone photoresponses

“…For example, cone visual pigment was purified and its quantum efficiency was shown to be similar to that of the rod pigment rhodopsin (11). In other studies, with the use of a purified cone protein, its interaction with a rod protein was measured (12)(13)(14)(15)(16). To know the mechanism characterizing the cone photoresponses, however, it is essential to measure the interaction between cone proteins and compare the result with the corresponding reactions in rods.…”

Light-dependent Activation of Rod Transducin by Pineal Opsin

“…What Determines the Number of Transducin Molecules Activated by P-opsin?-We have shown that P-opsin activates rod transducin in a light-dependent manner that is similar to rhodopsin but distinct from violet cone opsin (27). Moreover, Popsin has a meta-stable active conformation that is very similar to metarhodopsin II.…”

“…2) at 4°C. Membranes and 11-cis-retinal (final concentration, 5 M) were incubated overnight, solubilized by the addition of 10% (w/v) dodecyl maltoside (DM) to a final concentration of 1% DM, and incubated for 2 h. Insoluble material was removed by centrifugation at 40,000 ϫ g for 1 h. The P-opsin protein was purified on 1D4-Sepharose by immunoaffinity chromatography (26,27). Rhodopsin from bovine retina was purified in parallel with P-opsin.…”

“…Thus, P-opsin produces a meta-stable active state when exposed to light, with properties that parallel metarhodopsin II. In comparison to other opsins, P-opsin remains active 5 times longer than violet cone opsin (27) and its active state decays more slowly than that of green cone opsin (as estimated by spectroscopic measurements; Refs. 49 and 50).…”

“…Nucleotide exchange assays were performed using purified components under conditions that produce high activity of similarly prepared bovine rhodopsin (46) and violet cone opsin (27). Transducin activation by P-opsin was strictly light-dependent (i.e.…”