Molecular determinants of response kinetics of mouse M1 intrinsically-photosensitive retinal ganglion cells

Intrinsically-photosensitive retinal ganglion cells (ipRGCs) are non-rod/non-cone retinal photoreceptors expressing the visual pigment, melanopsin, to detect ambient irradiance for various non-image-forming visual functions. The M1-subtype, amongst the best studied, mediates primarily circadian photoentrainment and pupillary light reflex. Their intrinsic light responses are more prolonged than those of rods and cones even at the single-photon level, in accordance with the typically slower time course of non-image-forming vision. The short (OPN4S) and long (OPN4L) alternatively-spliced forms of melanopsin proteins are both present in M1-ipRGCs, but their functional difference is unclear. We have examined this point by genetically removing the Opn4 gene (Opn4(-/-)) in mouse and re-expressing either OPN4S or OPN4L singly in Opn4(-/-) mice by using adeno-associated virus, but found no obvious difference in their intrinsic dim-flash responses. Previous studies have indicated that two dominant slow steps in M1-ipRGC phototransduction dictate these cells' intrinsic dim-flash-response kinetics, with time constants (tau(1) and tau(2)) at room temperature of similar to 2 s and similar to 20 s, respectively. Here we found that melanopsin inactivation by phosphorylation or by beta-arrestins may not be one of these two steps, because their genetic disruptions did not prolong the two time constants or affect the response waveform. Disruption of GAP (GTPase-Activating-Protein) activity on the effector enzyme, PLC beta 4, in M1-ipRGC phototransduction to slow down G-protein deactivation also did not prolong the response decay, but caused its rising phase to become slightly sigmoidal by giving rise to a third time constant, tau(3) , of similar to 2 s (room temperature). This last observation suggests that GAP-mediated G-protein deactivation does partake in the flash-response termination, although normally with a time constant too short to be visible in the response waveform.

Automated summary

Intrinsically-photosensitive retinal ganglion cells (ipRGCs) express melanopsin to detect ambient irradiance for non-image-forming visual functions, with the M1-subtype primarily mediating circadian photoentrainment and pupillary light reflex. Studies have identified two slow steps in the intrinsic dim-flash-response kinetics of M1-ipRGCs, with no clear functional difference between the short and long alternatively-spliced forms of melanopsin proteins in these cells. Disruptions in melanopsin inactivation or G-protein deactivation did not significantly affect the response waveform in M1-ipRGCs.