Heterologous Expression of Melanopsin. II. Spectral Sensitivity and Phototransduction Cascade

Abstract

Abstract: : Purpose:Melanopsin is selectively expressed by intrinsically photosensitive retinal ganglion cells (ipRGCs) and is probably their photopigment. However, some have questioned whether melanopsin can form a functional photopigment or whether its absorption spectrum matches the spectral tuning of ipRGCs. Little is known about how melanopsin might gate the light–activated channels of ipRGCs. In a companion abstract (Kumbalasiri et al.), we show that heterologous expression of melanopsin confers photosensitivity upon a mammalian cell line. Here, we test the prediction that the spectral tuning of the heterologous system matches that of ipRGCs. We also analyzed the signaling cascade in the heterologous system for possible clues to phototransduction mechanisms in ipRGCs. Methods: Mouse melanopsin was heterologously expressed in HEK293–TRPC3 cells (see companion abstract) and light–evoked responses recorded by whole–cell methods. For spectral work, we provided 11–cis retinal and measured responses to narrow–band spectral lights. Drugs were applied through the bath or, for most G–protein blockers, through the pipette. Results: The action spectrum of the melanopsin–based photoresponse of HEK293–TRPC3 cells was virtually identical to that of ipRGCs. It closely fit a retinaldehyde template function and peaked at 479nm (cf. 484nm for rat ipRGCs). The phototransduction cascade in HEK293–TRPC3 cells resembled that in Drosophila photoreceptors. G–proteins of the Gq/11 family were implicated since GDPßS (a G–protein inhibitor) and GPant–2a (a Gq inhibitor) suppressed the response, but pertussis toxin (a Gi inhibitor) did not. Phospholipase C, the signaling enzyme coupled to Gq/11, was also implicated because a specific antagonist (U73122) strongly suppressed the photoresponse. TRPC3 formed the light–activated channel judging from the voltage dependence of the light–activated current, its suppression by non–selective blockers of TRPC channels (La³⁺; SK96365), and its absence in HEK293 cells lacking the TRPC3 protein. Conclusions: The spectral tuning of melanopsin–dependent photoresponses in these cells matches that in ipRGCs, strongly supporting the view that melanopsin is the ipRGC photopigment. In HEK293–TRPC3 cells, melanopsin couples to a phosphoinositide signaling cascade like that in many invertebrate (especially Drosophila) photoreceptors. A similar phototransduction cascade may (but need not) operate in ipRGCs.

Keywords: opsins • photoreceptors • circadian rhythms