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Title: Functional characterisation of key residues in the photopigment melanopsin
Author: Rodgers, Jessica
ISNI:       0000 0004 6495 8162
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Melanopsin (Opn4) is the opsin photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs). It has a conserved opsin structure and activation mechanism, yet demonstrates unusual functional properties that suggest it will possess unique structure-function relationships. The aim of this thesis was to characterise key OPN4 residues by examining the impact of non-synonymous mutations on melanopsin function. A genotype-driven screen of a chemically-mutagenized mouse archive led to the identification of a novel Opn4 mutant, S310A, located at a known opsin spectral tuning site. Action spectra from ipRGC and pupil light responses (PLR) of Opn4S310A mice revealed no change in wavelength of peak sensitivity. However, Opn4S310A PLR was significantly less sensitive at longer wavelengths, consistent with a short-wavelength shift in spectral sensitivity. This suggests S310A acts as a spectral tuning site in melanopsin. Next, the impact of naturally-occurring missense variants in human melanopsin (hOPN4) was examined in vitro. Fluorescent calcium imaging of 16 hOPN4 variants expressed in HEK293 cells revealed four hOPN4 variants abolished or attenuated responses to light (Y146C, R168C, G208S and S308F). These variants were located in conserved opsin motifs for chromophore binding or hydrogen-bond networks, functional roles apparently shared by melanopsin. Finally, two hOPN4 single nucleotide polymorphisms (SNPs) P10L and T394I, associated with abnormal non-image forming behaviour in humans, were explored in vivo. Using targeted viral-delivery of hOPN4 SNPs to mouse ipRGCs, a range of OPN4-driven behaviours, such as circadian photoentrainment and pupil light responses, were found to be comparable with hOPN4 WT control. Multi-electrode array recordings of ipRGCs transduced with hOPN4 T394I virus had significantly attenuated sensitivity and faster response offset, indicating this site may be functionally important for melanopsin activity but compensatory rod and cone input limits changes to non-image forming behaviour.
Supervisor: Hankins, Mark ; Peirson, Stuart Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Opsins ; Retina ; Biological Rhythms ; Optogenetics ; G-protein coupled receptor ; Opn4 ; Missense Mutation ; ipRGC ; Circadian Rhythms ; Adeno-associated virus ; Spectral Tuning ; Single Nucleotide Polymorphism ; Photopigment ; Melanopsin ; Opsin ; Pupillometry ; Multi-electrode array