Ectopic expression of two cone opsins in mouse RGCs results in opposite responses to light stimulation, likely due to differential G protein activation

Retinitis pigmentosa, a leading cause of inherited blindness, results in photoreceptor degeneration that current optogenetic approaches aim to address through microbial opsin expression in retinal ganglion cells (RGCs). While these microbial proteins restore light sensitivity, their clinical potential remains limited by low light sensitivity and immunogenicity risks. Recent efforts have focused on vertebrate opsins as safer alternatives, with mid-wavelength cone opsin (MW-opsin) demonstrating RGC depolarization via endogenous G-protein signaling. In this study, we reveal a surprising divergence in signaling outcomes of expressing short-wavelength mouse cone opsin (Opn1sw) in RGCs of blind mice. Using multielectrode array recordings and whole-cell patch clamping, we demonstrate that Opn1sw induces membrane hyperpolarization in RGCs – a stark contrast to MW-opsin’s depolarizing effects. This unexpected inversion suggests differential engagement of intracellular signaling pathways, potentially stemming from distinct G-protein coupling preferences. Comparative analysis of native G-protein expression profiles in RGCs versus cone photoreceptors supports this hypothesis, revealing mismatches that may explain ectopic opsin behavior. Our findings challenge the assumption of conserved opsin signaling across spectral subtypes and cell types, highlighting critical gaps in understanding vertebrate opsin-G protein interactions in non-native cellular environments. This discovery points to the necessity of systematic characterization of opsin signaling networks in target retinal cells, a prerequisite for engineering optimized optogenetic tools that reliably produce desired electrophysiological outcomes. By establishing spectral subtype-dependent signaling divergence, our work redefines parameters for developing next-generation vision restoration therapies.