Purchase this article with an account.
Jessica Rodgers, Steven Hughes, Carina A. Pothecary, Laurence A. Brown, Michael Parsons, Patrick Nolan, Stuart N. Peirson, Mark W. Hankins; Characterisation of a novel ENU-induced spectral tuning mutation in melanopsin. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5574.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Melanopsin (Opn4) is an opsin expressed in vertebrate photosensitive retinal ganglion cells. Several post-translation modification sites have been identified, but comparatively little is known about melanopsin's key functional residues. We aimed to identify novel gain- or loss-of-function mutations in melanopsin using a genotype-driven screen of a large murine mutant archive and characterise them in vitro and in vivo, to generate optogenetic tools, which could be optimised for a number of applications, including visual restoration therapies.
11328 n-ethyl-n-nitrosourea (ENU) mutagenized mice from MRC Harwell were screened for point mutations in exon 6 of melanopsin. One point mutation (S310A) was identified and investigated using bioinformatics. Immunocytochemistry and fluorescent calcium imaging were performed using heterologous expression of wild type (WT) and S310A Opn4 in Neuro-2A cells. Opn4S310A mice were recovered and backcrossed with retinally degenerate (C3H/HeH- Pde6brd1) animals. Their non-image forming responses to 550nm light were assessed using pupillometry, where the light intensity (log quanta) required to produce half maximal pupil constriction (IC50) was used as a measure of sensitivity.
The ENU archive screen identified a serine to alanine substitution at position 310 (S310A) in transmembrane helix 6 of melanopsin. Amino acid alignments demonstrate S310A occurs at a homologous opsin spectral tuning site and represents a highly conserved residue difference between the X and M melanopsin gene classes. S310A Opn4 expressed in Neuro-2A cells is localised in the plasma membrane and produces an intracellular calcium increase upon light exposure, similar to WT Opn4. Pupil constriction in Opn4S310A (Mean IC50=13.55, SD=0.25, N=5) is significantly attenuated at 550nm compared to Opn4WT controls (Mean IC50=13.05, SD=0.12, N=4) compared using a two-tailed Student’s t-test, t(7) = -3.59, p = 0.009. This IC50 difference is consistent with a 14nm hypsochromic shift in the spectral sensitivity of S310A Opn4.
We have identified a putative spectral tuning mutant, S310A Opn4. This forms a functional photopigment, capable of coupling to a Gq/11 signalling pathway. Pupillometry data suggests S310A Opn4 may be spectrally shifted to shorter wavelengths, consistent with evidence that mutation of the equivalent position in other opsins causes hypsochromic shifts in spectral tuning.
This PDF is available to Subscribers Only