Abstract
Purpose:
Opsins are light-sensitive G-protein coupled receptor proteins, essential for vision, circadian rhythmicity and eye development. Opsins function by activating G-protein second messenger systems. Rod and cone opsins activate Gαt, while melanopsin activates Gαq/11. If inner retinal neurons, such as bipolar cells, were engineered to become light sensitive, these cells could act as substitute photoreceptors in patients who have lost their photoreceptors. We have fused human melanopsin to different Gα-proteins to test whether such a fusion modifies the coupling of an opsin to a G-protein second messenger system. An opsin-Gα-protein fusion could provide an optogentic tool for restoring sight in humans.
Methods:
Two melanopsin-Gα-protein fusion constructs were cloned into pMT4 by removing the stop codon from melanopsin and inserting the in-frame coding sequence of either GNAQ (encoding Gαq) or GNA11 (Gα11). Calcium kinetics were observed using Rhod-2 fluorescent dye. Small interfering RNAs (siRNA) targeting endogenous Gα-protein transcripts (including GNAQ and GNA11) were applied to HEK293T cells expressing wild type melanopsin and melanopsin-Gα-protein fusions to determine the relative importance of the fused Gα-protein to activating the intracellular signalling cascade.
Results:
Melanopsin-Gαq/Gα11 fusion proteins exhibited a similar response rate (41% and 40%, respectively) and time course of calcium kinetics compared to non-fused melanopsin (48%; no statistically significant differences between groups on ANOVA with post hoc Tukey HSD). Using siRNA to knock down endogenous levels of Gα-proteins in HEK293T cells showed melanopsin-Gα-protein fusion transfected cells to have a higher response rate than wild type melanopsin transfected cells (melanopsin-Gα11 response rate 36%, wild type melanopsin 13%, p>0.05).
Conclusions:
Fusing melanopsin to either of its native Gα subunits, Gαq or Gα11, shows that melanopsin can maintain coupling to the Gαq/11 second messenger system in the presence of a fused Gαq or Gα11 subunit. Furthermore, transient expression of melanopsin-Gα protein fusions in HEK293T cells with siRNA-induced knock down of endogenous Gα subunits suggests that fusing a Gα subunit to melanopsin enables greater efficiency of coupling to the second messenger pathway. Melanopsin-Gα protein constructs may therefore offer advantages over wild type melanopsin as a potential optogenetic gene therapy for photoreceptor loss.
Keywords: 625 opsins •
710 second messengers •
659 protein structure/function