In conclusion, our data collectively suggest that the residual GC activity induced by the cone-rod dystrophy-associated G86R GCAP1 mutant under high Ca2+ may be caused by the intramolecular lock constituted by the R86-W21 and R86-W94 cation-π interactions, which ultimately prevent the correct structural rearrangement necessary for promoting the activator to inhibitor conformational transition. Here, GUCA1A is linked to cone-rod dystrophy.