Functional analysis of two novel retinitis pigmentosa mutations reveals structural role of the third transmembrane helix in rhodopsin photoactivation.

Mutations in the visual receptor rhodopsin are a common cause of inherited retinal diseases. Many of these mutations occur within the transmembrane core of the receptor and can impact in its folding and function. In this study, we investigate two mutations associated with the retinal degenerative disease retinitis pigmentosa, T1083.23P and G1213.36R, both located in the third transmembrane helix of rhodopsin and in proximity to key structural residues essential for rhodopsin stability and proper folding. We find that the G1213.36R mutant fails to regenerate with 11-cis-retinal, suggesting that the mutation severely impairs chromophore binding and proper folding. In contrast, the T1083.23P mutant regenerates efficiently, shows normal photobleaching and acidification profiles, and similar chromophore regeneration to that of the wild-type protein. However, the T1083.23P mutation leads to reduced thermal and chemical stability of its dark state, and a delayed formation of the active conformation. These conformational alterations correlate with a slower and less efficient activation of transducin, indicating that T1083.23P partially decouples the light-induced response from downstream G-protein signaling. Together, these findings demonstrate that amino acid substitutions in the third transmembrane helix of rhodopsin can have diverse molecular consequences: from the severe impairment of chromophore binding in G1213.36R to selective destabilization and signaling defects in the T1083.23P case. Our results emphasize the importance of the third transmembrane helix in supporting the conformational changes necessary for rhodopsin activation. and contribute to a deeper understanding of how specific mutations in rhodopsin may elicit distinct pathogenic mechanisms underlying retinal degeneration.