Functional characterization of the rod visual pigment of the echidna (Tachyglossus aculeatus), a basal mammal

Abstract

Monotremes are the most basal egg-laying mammals comprised of two extant genera, which are largely nocturnal. Visual pigments, the first step in the sensory transduction cascade in photoreceptors of the eye, have been examined in a variety of vertebrates, but little work has been done to study the rhodopsin of monotremes. We isolated the rhodopsin gene of the nocturnal short-beaked echidna (Tachyglossus aculeatus) and expressed and functionally characterized the protein in vitro. Three mutants were also expressed and characterized: N83D, an important site for spectral tuning and metarhodopsin kinetics, and two sites with amino acids unique to the echidna (T158A and F169A). The k max of echidna rhodopsin (497.9 6 1.1 nm) did not vary significantly in either T158A (498.0 6 1.3 nm) or F169A (499.4 6 0.1 nm) but was redshifted in N83D (503.8 6 1.5 nm). Unlike other mammalian rhodopsins, echidna rhodopsin did react when exposed to hydroxylamine, although not as fast as cone opsins. The retinal release rate of light-activated echidna rhodopsin, as measured by fluorescence spectroscopy, had a half-life of 9.5 6 2.6 min À1 , which is significantly shorter than that of bovine rhodopsin. The half-life of the N83D mutant was 5.1 6 0.1 min À1 , even shorter than wild type. Our results show that with respect to hydroxylamine sensitivity and retinal release, the wild-type echidna rhodopsin displays major differences to all previously characterized mammalian rhodopsins and appears more similar to other nonmammalian vertebrate rhodopsins such as chicken and anole. However, our N83D mutagenesis results suggest that this site may mediate adaptation in the echidna to dim light environments, possibly via increased stability of light-activated intermediates. This study is the first characterization of a rhodopsin from a most basal mammal and indicates that there might be more functional variation in mammalian rhodopsins than previously assumed.

Cone-like rhodopsin expressed in the all cone retina of the colubrid pine snake as a potential adaptation to diurnality

“…2B), which is similar to the measured λ max of rhodopsins from T. proximus, T. sirtalis and Arizona elegans snakes (Schott et al, 2016;Sillman et al, 1997;Simões et al, 2016). The drastic blue shift is expected given the presence of the blue-shifting N83 and S292 amino acid identities (Bickelmann et al, 2012;Dungan et al, 2016;van Hazel et al, 2016). Pituophis melanoleucus rhodopsin expression was similar to that of T. proximus, with a large ratio between total purified protein (absorbance at 280 nm) and active protein (absorbance at λ max ) that indicates that only a small proportion of the translated opsin protein is able to bind chromophore and become functionally active.…”

“…With a reaction half-life of ∼14 min, the P. melanoleucus rhodopsin reacts much quicker and closer to cone opsin speeds (Das et al, 2004;Ma et al, 2001) than previous rhodopsins that have reacted when incubated in hydroxylamine, such as those of the echidna (Bickelmann et al, 2012) and the anole (Kawamura and Yokoyama, 1998), which react over hours. The RH1 sequence contains both E122 and I189, which are known to mediate the slower decay and regeneration kinetics typical of rhodopsin (Imai et al, 1997;Kuwayama et al, 2002).…”

“…Mutations at site 185 have been shown to reduce both visual pigment stability (McKibbin et al, 2007) and transducin activation in vitro (Karnik et al, 1988). Also, the P. melanoleucus RH1 has N83 and S292, which are often found in rhodopsins with blue-shifted λ max values, and can also affect all-trans retinal release kinetics following photoactivation (Bickelmann et al, 2012;van Hazel et al, 2016).…”

Comparative Mutagenesis Studies of Retinal Release in Light-Activated Zebrafish Rhodopsin Using Fluorescence Spectroscopy

“…One of the few studies to examine retinal release in a nonmodel organism, the echidna, suggests that variation in kinetic rates exists among mammalian rhodopsins, 23 even if the differences are more subtle than those between rhodopsin and cone opsins. 20 This is not surprising considering that other functional differences among rhodopsins have also been reported, including hydroxylamine stability, 24 and kinetics of metarhodopsin intermediates.…”

The evolution of opsins and color vision: connecting genotype to a complex phenotype

“…In these assays cultured primate or human cells are used to express opsin proteins that are then reconstituted with their corresponding chromophore, either A1 or A2 (Fig 3). This process produces functional, purified visual pigments in enough quantity to measure their spectral sensitivity (Chang, 2003;Bloch et al, 2015a;Bloch et al, 2015b) and more recently other less understood functions (Bickelmann et al, 2012;Morrow and Chang, 2015). In addition to studying the function of extant visual pigments, these assays can be combined with site-directed mutagenesis in order to recreate and express ancestral visual pigments, thus providing a way to trace the evolution of genes and function all the way from a clade's ancestor (Chang, 2003;Bloch et al, 2015a) and to study to role of specific substitutions on spectral tuning (Yokoyama and Radlwimmer, 2001;Yokoyama et al, 2008a).…”