Functional characterisation of key residues in the photopigment melanopsin
<p>Melanopsin (<em>Opn4</em>) 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-functio...
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| Format: | Thesis |
| Language: | English |
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2016
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| _version_ | 1797096260855922688 |
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| author | Rodgers, J |
| author2 | Hankins, M |
| author_facet | Hankins, M Rodgers, J |
| author_sort | Rodgers, J |
| collection | OXFORD |
| description | <p>Melanopsin (<em>Opn4</em>) 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 <em>OPN4</em> 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 <em>Opn4</em> mutant, S310A, located at a known opsin spectral tuning site. Action spectra from ipRGC and pupil light responses (PLR) of <em>Opn4<sup>S310A</sup></em> mice revealed no change in wavelength of peak sensitivity. However, <em>Opn4<sup>S310A</sup></em> 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 (<em>hOPN4</em>) was examined <em>in vitro</em>. Fluorescent calcium imaging of 16 <em>hOPN4</em> variants expressed in HEK293 cells revealed four <em>hOPN4</em> 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 <em>hOPN4</em> 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 <em>hOPN4</em> SNPs to mouse ipRGCs, a range of <em>OPN4</em>-driven behaviours, such as circadian photoentrainment and pupil light responses, were found to be comparable with <em>hOPN4</em> WT control. Multi-electrode array recordings of ipRGCs transduced with <em>hOPN4</em> 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.</p> |
| first_indexed | 2024-03-07T04:39:21Z |
| format | Thesis |
| id | oxford-uuid:d1184150-9b61-4cc9-94ad-2cc13a3d21ce |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T04:39:21Z |
| publishDate | 2016 |
| record_format | dspace |
| spelling | oxford-uuid:d1184150-9b61-4cc9-94ad-2cc13a3d21ce2022-03-27T07:54:36ZFunctional characterisation of key residues in the photopigment melanopsinThesishttp://purl.org/coar/resource_type/c_db06uuid:d1184150-9b61-4cc9-94ad-2cc13a3d21ceRetinaOptogeneticsOpsinsBiological RhythmsEnglishORA Deposit2016Rodgers, JHankins, MPeirson, S<p>Melanopsin (<em>Opn4</em>) 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 <em>OPN4</em> 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 <em>Opn4</em> mutant, S310A, located at a known opsin spectral tuning site. Action spectra from ipRGC and pupil light responses (PLR) of <em>Opn4<sup>S310A</sup></em> mice revealed no change in wavelength of peak sensitivity. However, <em>Opn4<sup>S310A</sup></em> 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 (<em>hOPN4</em>) was examined <em>in vitro</em>. Fluorescent calcium imaging of 16 <em>hOPN4</em> variants expressed in HEK293 cells revealed four <em>hOPN4</em> 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 <em>hOPN4</em> 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 <em>hOPN4</em> SNPs to mouse ipRGCs, a range of <em>OPN4</em>-driven behaviours, such as circadian photoentrainment and pupil light responses, were found to be comparable with <em>hOPN4</em> WT control. Multi-electrode array recordings of ipRGCs transduced with <em>hOPN4</em> 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.</p> |
| spellingShingle | Retina Optogenetics Opsins Biological Rhythms Rodgers, J Functional characterisation of key residues in the photopigment melanopsin |
| title | Functional characterisation of key residues in the photopigment melanopsin |
| title_full | Functional characterisation of key residues in the photopigment melanopsin |
| title_fullStr | Functional characterisation of key residues in the photopigment melanopsin |
| title_full_unstemmed | Functional characterisation of key residues in the photopigment melanopsin |
| title_short | Functional characterisation of key residues in the photopigment melanopsin |
| title_sort | functional characterisation of key residues in the photopigment melanopsin |
| topic | Retina Optogenetics Opsins Biological Rhythms |
| work_keys_str_mv | AT rodgersj functionalcharacterisationofkeyresiduesinthephotopigmentmelanopsin |