Constitutive activity of a UV cone opsin

Abstract

Vertebrate visual pigment proteins contain a conserved carboxylic acid residue in the third transmembrane helix. In rhodopsin, Glu113 serves as a counterion to the positively charged protonated Schiff base formed by 11-cis retinal attached to Lys296. Activation involves breaking of this ion pair. In UV cone pigments, the retinyl Schiff base is unprotonated, and hence such a salt bridge is not present; yet the pigment is inactive in the dark. Mutation of Glu108, which corresponds to rhodopsin's Glu113, to Gln yields a pigment that remains inactive in the dark. The apoproteins of both the wild-type and mutant, however, are constitutively active with the mutant being of significantly higher activity. Thus, one important role for preserving the negatively charged glutamate in the third helix of UV pigments is to maintain a less active opsin in a manner similar to rhodopsin. Ligand binding itself in the absence of a salt bridge is sufficient for deactivation.

Figure 1

Absorption spectra of wild-type (thin black lines) and E108Q mutant (thick black lines) salamander UV cone pigment in the dark and acid-denatured forms. Spectra were normalized to the acid-denatured species.

Figure 2

Transducin activation by purified wild-type (triangles) and E108Q mutant (circles) salamander UV cone pigment. Both pigments are able to activate transducin in a light-dependent manner. Note the identical lack of activation by the pigments in the dark. The reaction mixtures both contained 10 nM pigment as determined from the acid-denatured spectrum of the samples from Figure 1.

Figure 3

Transducin activation by (A) E108Q mutant and (B) wild-type salamander UV cone opsins (open symbols) and pigments (filled symbols). To the opsin sample, 1 μL ethanol was added; to the pigment sample, 1 μL 11-cis retinal in ethanol was added to a final concentration of 240 μM and incubated at least 1 h. Error bars reflect standard deviation of the means for both activity and opsin concentration.