July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Chimeric human opsins as optogenetic light sensitizers
Author Affiliations & Notes
  • Doron Hickey
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
    Royal Victorian Eye and Ear Hospital, Heidelberg, Victoria, Australia
  • Wayne IL Davies
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
    School of Biological Sciences and UWA Oceans Institute, Perth, Western Australia, Australia
  • Steven Hughes
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
    Sleep and Circadian Neuroscience Institute, University of Oxford, Oxford, United Kingdom
  • Jessica Rodgers
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
    Division of Neuroscience and Experimental Psychology, University of Manchester, Manchester, United Kingdom
  • Navamayooran Thavanesan
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
  • Robert E MacLaren
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
    Oxford University Hospitals NHS Trust Biomedical Research Centre, Oxford, United Kingdom
  • Mark W Hankins
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
    Sleep and Circadian Neuroscience Institute, University of Oxford, Oxford, United Kingdom
  • Footnotes
    Commercial Relationships   Doron Hickey, None; Wayne Davies, None; Steven Hughes, None; Jessica Rodgers, None; Navamayooran Thavanesan, None; Robert MacLaren, None; Mark Hankins, None
  • Footnotes
    Support  Woolf Fisher Trust, Wellcome Trust, BBSRC, NIHR Biomedical Research Centres of Oxford and Moorfields, MRC
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 5989. doi:
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      Doron Hickey, Wayne IL Davies, Steven Hughes, Jessica Rodgers, Navamayooran Thavanesan, Robert E MacLaren, Mark W Hankins; Chimeric human opsins as optogenetic light sensitizers. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5989.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Human opsin-based photopigments have great potential as light-sensitizers, but their requirement for phototransduction cascade-specific second messenger proteins limits their functionality in non-native cell types. Human chimeric opsins where intracellular domains of representative visual opsins were replaced by corresponding regions of Gq/11-coupled melanopsin were investigated as potential optogenetic tools.

Methods : Eight chimeric human opsins were generated that consisted of either a human rod opsin or long-wavelength-sensitive opsin (LWS) backbone with intracellular domains that were replaced with corresponding regions derived from human melanopsin. Opsin protein trafficking and Gi and Gq/11 second messenger coupling were investigated by immunocytochemistry, and cAMP and calcium live-cell assays. Chromophore requirements were also investigated.

Results : Rhodopsin/melanopsin chimeric pigments were able to couple to both Gq/11 and Gi pathways to varying degrees: greater substitution of the intracellular surface with corresponding melanopsin domains generally showed greater Gq/11 activity with a decrease in the ability to activate Gi (area under the curve (AUC) analysis for each dataset: P < 0.0001, ordinary one-way ANOVA; n = 11 and 8, respectively). The relative time to peak Gq/11 fluorescence showed statistically significant differences between all groups (F (6, 62) = 6.4, P < 0.0001, ordinary one-way ANOVA; n = 11). Unlike melanopsin, rhodopsin and rhodopsin/melanopsin chimeric opsins were found to require exogenous 9-cis retinal to function. Wild type LWS opsin and LWS/melanopsin chimeras showed only minor Gi activation (F (6, 21) = 0.274, P = 0.142, ordinary one-way ANOVA; n = 4) and no Gq/11 activity in response to light stimulation. Immunocytochemistry demonstrated that chimeric opsins with more intracellular domains derived from melanopsin were less likely to be trafficked to the plasma membrane and more likely to lead to the formation of aggresomes.

Conclusions : By creating these unique human opsins that combine the spectral and chromophore requirements of rhodopsin with the intracellular signaling properties of melanopsin, this study demonstrates the importance of Gα coupling efficiency to the speed of cellular responses, and created human opsins with a unique combination of properties to expand the range of customised optogenetic biotools for basic research and translational therapies.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

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