The C-Terminus and Third Cytoplasmic Loop Cooperatively Activate Mouse Melanopsin Phototransduction

Area of Science:

• Optogenetics and Photobiology
• Molecular and Cellular Biology
• Neuroscience

Background:

• Melanopsin mediates non-image-forming visual responses like circadian rhythms and pupil reflexes.
• Intrinsically photosensitive retinal ganglion cells (ipRGCs) exhibit slow light response kinetics.
• While melanopsin deactivation is understood, its activation mechanism remains unclear.

Purpose of the Study:

• To investigate the molecular determinants of mouse melanopsin activation.
• To elucidate the structural interactions governing melanopsin's inactive conformation and activation process.
• To determine the role of C-terminal phosphorylation in melanopsin signaling.

Main Methods:

• Site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy.
• Calcium imaging of melanopsin mutants.
• Comparative analysis with armadillo melanopsin.

Main Results:

• EPR data suggest steric freedom in the third intracellular loop, increasing upon C-terminus truncation.
• Mutations affecting the proximal C-terminus (truncation, proline substitutions) and phosphorylation sites (including Y382) significantly delayed melanopsin activation.
• Armadillo melanopsin mutations also showed slower activation, supporting the role of phosphorylation.

Conclusions:

• Mouse melanopsin's C-terminus is spatially close to intracellular loop 3 in the inactive state.
• C-terminal phosphorylation facilitates an ionic interaction, stabilizing the structure for G-protein signaling initiation.
• This structural conformation is critical for regulating melanopsin activation kinetics.