Single‐Residue Substitution of Opicalcin1 Enhances Cellular Uptake but Reduces Ca 2+ ‐Release Activity

The Calcin family comprises scorpion venom‐derived peptides with cell‐penetrating activity and reported modulatory effects on RyR‐associated Ca 2+ signaling. OpiCa1 differs from its homolog OpiCa2 at residue 26, making this position a candidate site for examining structure–activity relationships related to cellular uptake and Ca 2+ ‐release activity. To assess the contribution of residue 26, we generated two OpiCa1 variants, T26R and T26K by replacing Thr26 with the basic residues arginine and lysine. We compared these variants with parental OpiCa1 using in silico modeling, fluorescence‐based uptake assays, cytotoxicity assessment, and Ca 2+ imaging. Among the tested variants, T26R showed significantly enhanced cellular uptake compared with parental OpiCa1 and exhibited no detectable cytotoxicity at 10–60 µM under the tested conditions. In silico analyses suggested possible changes in the predicted interaction mode of T26R near the RyR S6 region. Consistent with these computational observations, cellular functional assays revealed that T26R, despite its superior uptake, concurrently attenuated Ca 2+ ‐release activity. These findings suggest that residue 26 contributes to the cellular uptake and Ca 2+ ‐release activity of OpiCa1, while further experimental studies are required to define its direct interaction with RyRs.