Light control of G protein signaling pathways by a novel photopigment.

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.

Characterization of opsin gene alleles affecting color vision in a wild population of titi monkeys (Callicebus brunneus).

The color vision of most platyrrhine primates is determined by alleles at the polymorphic X-linked locus coding for the opsin responsible for the middle- to long-wavelength (M/L) cone photopigment. Females who are heterozygous at the locus have trichromatic vision, whereas homozygous females and all males are dichromatic. This study characterized the opsin alleles in a wild population of the socially monogamous platyrrhine monkey Callicebus brunneus (the brown titi monkey), a primate that an earlier study suggests may possess an unusual number of alleles at this locus and thus may be a subject of special interest in the study of primate color vision. Direct sequencing of regions of the M/L opsin gene using feces-, blood-, and saliva-derived DNA obtained from 14 individuals yielded evidence for the presence of three functionally distinct alleles, corresponding to the most common M/L photopigment variants inferred from a physiological study of cone spectral sensitivity in captive Callicebus.

Retinal photoreceptor arrangement, SWS1 and LWS opsin sequence, and electroretinography in the South American marsupial Thylamys elegans (Waterhouse, 1839).

We studied the retinal photoreceptors in the mouse opossum Thylamys elegans, a nocturnal South American marsupial. A variety of photoreceptor properties and color vision capabilities have been documented in Australian marsupials, and we were interested to establish what similarities and differences this American marsupial showed. Thylamys opsin gene sequencing revealed two cone opsins, a longwave-sensitive (LWS) opsin and a shortwave-sensitive (SWS1) opsin with deduced peak sensitivities at 560 nm and 360 nm (ultraviolet), respectively. Immunocytochemistry located these opsins to separate cone populations, a majority of LWS cones (density range 1,600-5,600/mm(2)) and a minority of SWS1 cones (density range 100-690/mm(2)). With rod densities of 440,000-590,000/mm(2), the cones constituted 0.4-1.2% of the photoreceptors. This is a suitable adaptation to nocturnal vision. Cone densities peaked in a horizontally elongated region ventral to the optic nerve head. In ventral-but not dorsal-retina, roughly 40% of the LWS opsin-expressing cones occurred as close pairs (double cones), and one member of each double cone contained a colorless oil droplet. The corneal electroretinogram (ERG) showed a high scotopic sensitivity with a rod peak sensitivity at 505 nm. At mesopic light levels, the spectral ERG revealed the contributions of a UV-sensitive SWS1 cone mechanism and an LWS cone mechanism with peak sensitivities at 365 nm and 555 nm, respectively, confirming the tuning predictions from the cone opsin sequences. The two spectral cone types provide the basis for dichromatic color vision, or trichromacy if the rods contribute to color processing at mesopic light levels.