Abstract
Channelopsins and photo-regulated ion channels make it possible to use light to control electrical activity of cells. This powerful approach has lead to a veritable explosion of applications, though it is limited to changing membrane voltage of the target cells. An enormous potential could be tapped if similar opto-genetic techniques could be extended to the control of chemical signaling pathways. Photopigments from invertebrate photoreceptors are an obvious choice—as they do not bleach upon illumination -however, their functional expression has been problematic. We exploited an unusual opsin, pScop2, recently identified in ciliary photoreceptors of scallop. Phylogenetically, it is closer to vertebrate opsins, and offers the advantage of being a bi-stable photopigment. We inserted its coding sequence and a fluorescent protein reporter into plasmid vectors and demonstrated heterologous expression in various mammalian cell lines. HEK 293 cells were selected as a heterologous system for functional analysis, because wild type cells displayed the largest currents in response to the G-protein activator, GTP-γ-S. A line of HEK cells stably transfected with pScop2 was generated; after reconstitution of the photopigment with retinal, light responses were obtained in some cells, albeit of modest amplitude. In native photoreceptors pScop2 couples to Go; HEK cells express poorly this G-protein, but have a prominent Gq/PLC pathway linked to internal Ca mobilization. To enhance pScop2 competence to tap into this pathway, we swapped its third intracellular loop—important to confer specificity of interaction between 7TMDRs and G-proteins—with that of a Gq-linked opsin which we cloned from microvillar photoreceptors present in the same retina. The chimeric construct was evaluated by a Ca fluorescence assay, and was shown to mediate a robust mobilization of internal calcium in response to illumination. The results project pScop2 as a potentially powerful optogenetic tool to control signaling pathways.
Highlights
Controlling cellular activity by exogenous stimulation can help unravel the functioning of cell ensembles and the neural control of behavior, and holds great promise for therapeutic intervention
Having recently cloned an ortholog in the related species, Pecten irradians and demonstrated that it mediates the light-response in ciliary photoreceptors [27], we assayed heterologous expression by lipofectamine transfection of this new member of the group in several mammalian cell lines (N2A, HEK292, CHO, and LLC-PK1CL4T), in combination with two expression vectors that encoded pScop2 in a fusion construct with either GFP or mRFP
The Gqcoupled receptor that served as donor was the opsin of the microvillar photoreceptors that comprise the proximal layer of the double retina of Pecten, and which are known to respond to light mobilizing Ca via the Gq/PLCβ pathway [38]. We cloned such opsin by PCR: having amplified a product homologous to the putative R-opsin previously identified in the Japanese scallop [26], we extended it by RACE (Fig 4A), obtaining overlapping amplicons that, upon CAP assembly, spanned 2168 bp, comprising a full-length transcript (Genebank accession number MG674156)
Summary
Controlling cellular activity by exogenous stimulation can help unravel the functioning of cell ensembles and the neural control of behavior, and holds great promise for therapeutic intervention. Even for controlling the electrical activity of the target cells, Gprotein pathways can be enlisted to exert a wide spectrum of modulatory influences on ion channels, altering, for example, open times [10] or inactivation [11] This general goal could be attained by utilizing an exogenously implanted 7-transmembrane receptor (7TMDR), whose activity could be controlled by light. Ingenious efforts in this direction have surfaced, like using a metabotropic glutamate receptor conjugated to an azobenzene-derived photoactivatable linker to which an agonist molecule has been attached: light-induced conformational transitions of the linker bring the agonist moiety close to or far from its binding site, allowing reversible light control of the receptor and its cognate G-protein pathway [12] This strategy is powerful, but complex: because neither the linker nor the agonist are proteic, they are introduced after expression of the suitably modified 7TMDR, which typically incorporates engineered cysteines to serve as acceptor of the linker-agonist complex via thiol chemistry. These additional steps reduce the generality and practical applicability of such approach
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