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

Published online by Cambridge University Press:  09 July 2012

CONSTANZE BICKELMANN ,
JAMES M. MORROW ,
JOHANNES MÜLLER  and
BELINDA S.W. CHANG
CONSTANZE BICKELMANN*
Affiliation:
Museum für Naturkunde – Leibniz-Institut für Evolutions- und Biodiversitätsforschung an der Humboldt-Universität zu Berlin, Berlin, Germany Present address: Paläontologisches Institut und Museum, Universität Zürich, Karl-Schmid-Strasse 4, 8006 Zurich, Switzerland
JAMES M. MORROW
Affiliation:
Department of Cell & Systems Biology, University of Toronto, Toronto, Ontario, Canada
JOHANNES MÜLLER
Affiliation:
Museum für Naturkunde – Leibniz-Institut für Evolutions- und Biodiversitätsforschung an der Humboldt-Universität zu Berlin, Berlin, Germany
BELINDA S.W. CHANG*
Affiliation:
Department of Cell & Systems Biology, University of Toronto, Toronto, Ontario, Canada Department of Ecology & Evolutionary Biology, University of Toronto, Toronto, Ontario, Canada Centre for the Analysis of Genome Evolution & Function, University of Toronto, Toronto, Ontario, Canada
*
†Address correspondence and reprint requests to: Dr. Constanze Bickelmann, Paläontologisches Institut und Museum, Universität Zürich, Karl-Schmid-Strasse 4, 8006 Zurich, Switzerland. E-mail: constanze.bickelmann@pim.uzh.ch and Prof. Belinda S.W. Chang, Department of Cell & Systems Biology, University of Toronto, 25, Harbord St., Toronto, Ontario, Canada M5S 3G5. E-mail: belinda.chang@utoronto.ca
†Address correspondence and reprint requests to: Dr. Constanze Bickelmann, Paläontologisches Institut und Museum, Universität Zürich, Karl-Schmid-Strasse 4, 8006 Zurich, Switzerland. E-mail: constanze.bickelmann@pim.uzh.ch and Prof. Belinda S.W. Chang, Department of Cell & Systems Biology, University of Toronto, 25, Harbord St., Toronto, Ontario, Canada M5S 3G5. E-mail: belinda.chang@utoronto.ca
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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 λmax of echidna rhodopsin (497.9 ± 1.1 nm) did not vary significantly in either T158A (498.0 ± 1.3 nm) or F169A (499.4 ± 0.1 nm) but was redshifted in N83D (503.8 ± 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 ± 2.6 min−1, which is significantly shorter than that of bovine rhodopsin. The half-life of the N83D mutant was 5.1 ± 0.1 min−1, 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.

Keywords

RhodopsinMammalsMonotremesRetinal release rateN83D mutagenesis
Type
Research Articles
Information
Visual Neuroscience , Volume 29 , Issue 4-5 , September 2012 , pp. 211 - 217
Copyright
Copyright © Cambridge University Press 2012

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