PHOTO-TRANSDUCTION MECHANISMS IN M2- AND M4-INTRINSICALLY PHOTOSENSITIVE RETINAL GANGLION CELLS

Non-image-forming vision in mammals is mediated primarily by melanopsin (OPN4)-expressing, intrinsically-photosensitive retinal ganglion cells (ipRGCs). In mouse M1-subtype ipRGCs, our laboratory has previously shown that melanopsin activates PLCβ4 (phospholipase C-β4) to open TRPC6,7 channels. Whether other ipRGC subtypes also adopt the same phototransduction mechanism still remains unknown. We have now examined mouse M2- and M4-cells. Surprisingly, we found a hitherto undescribed, melanopsin-driven ciliary phototransduction mechanism involving cyclic nucleotide as the second messenger in M2- and M4-ipRGCs. However, the last step of this phototransduction mechanism consists of the activation of an HCN (Hyperpolarization- and Cyclic-Nucleotide-gated) channel instead of a CNG (Cyclic-Nucleotide-Gated) channel as found in rods and cones and literally all other ciliary photoreceptors. The light-triggered generation of cyclic nucleotide in this rather unique pathway was verified by using a CNG (CNGA2) channel expressed heterologously via AAV2-virus in ipRGCs. Later experiments with photo-uncaging of caged-cyclic nucleotides has proven that the specific second messenger is cAMP, instead of cGMP as found for nearly all ciliary phototransduction mechanisms across the animal kingdom except jellyfish, which uses cAMP and is an evolutionarily ancient species. This second phototransduction pathway is also Gαq-subfamily-dependent. However, after knocking out Gαq-subfamily (Gαq, Gα11, and Gα14), the null-phenotype of the second pathway takes a longer time to develop than that of the M1-pathway. This suggests that the two pathways probably bifurcate at the G-protein step. Possibly, the second pathway is mediated by the Gβγ subunits that are coupled to Gαq-subfamily. The production of cAMP can arise from adenylyl cyclase activated directly by Gβγ subunits. Furthermore, we found that each M2-ipRGC has both the microvillous-motif PLC-mediated and the ciliary-motif cyclic-nucleotide-mediated pathways operating in parallel and synergistically. Our findings reveal a complex heterogeneity in phototransduction among ipRGCs and, more importantly, break a general dogma about segregation of the two phototransduction motifs. The co-existence of microvillous and ciliary phototransductions in a given cell, together with the involvement of cAMP instead of cGMP, may suggest that ipRGCs are extant ancestral photoreceptors in the animal kingdom