Heterologous Expression of Melanopsin. I. Induced Photosensitivity

Abstract

Abstract: : Purpose: Melanopsin has been proposed as the photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs). It is selectively expressed in these cells and is required for their photosensitivity. However, there is little direct evidence that melanopsin forms a functional photopigment, and some have questioned whether it can do so. We now demonstrate melanopsin’s photosensory capacity in a heterologous expression system. Methods: We transiently expressed mouse melanopsin in human embryonic kidney cells that stably expressed TRPC3 channels (HEK293–TRPC3). Full–length mouse melanopsin cDNA was PCR amplified and cloned into a bicistronic expression vector (pIRES2–EGFP), so that transfected cells would coexpress the EGFP marker protein and melanopsin. Cells were transfected by the calcium phosphate method and provided with all–trans or 11–cis retinaldehyde. Photosensitivity was tested by whole–cell patch recordings and by calcium imaging using the dye Rhod–2–AM. Results: Coexpression of melanopsin and EGFP was verified by confocal imaging of transfected cells after immunolabeling with an antibody to mouse melanopsin. Melanopsin was localized to the plasma membrane, as it is in ipRGCs. Most melanopsin–expressing HEK293–TRPC3 cells were photosensitive. Light evoked tonic, sluggish depolarizations and inward currents resembling those in ipRGCs. The light–evoked current reversed near 0 mV and exhibited strong outward rectification, consistent with its mediation by TRPC3 channels. Light also evoked substantial increases in intracellular calcium. Light responses were clearly dependent on the presence of the opsin because they were detectable in cells that expressed melanopsin without EGFP (pcDNA 3.1 vector), but not in cells expressing EGFP without melanopsin (empty pIRES2–EGFP vector) or in untransfected cells. Conclusions: Melanopsin heterologously expressed in HEK293–TRPC3 cells confers photosensitivity upon them. In a companion abstract (Qiu et al.), we show that the spectral tuning of this response also closely matches that of the photopigment in ipRGCs. We conclude that melanopsin can form a functional photopigment and that it is the photopigment of ganglion–cell photoreceptors.

Keywords: opsins • circadian rhythms • photoreceptors