Engineering of genetically encoded programmable calcium channel inhibitory binders

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels, composed of STIM and ORAI, are essential for immune and developmental processes, and their dysregulation underlies channelopathies such as Stormorken syndrome. Here, we report the engineering of genetically encoded CRAC channel inhibitory binders (CRABs) derived from the ORAI C-terminal tail. Guided by deep mutational scanning, we optimize a membrane-anchored CRAB variant that potently inhibits Ca2+ influx and NFAT signaling, and rescues thrombocytopenia-like phenotypes in a zebrafish model of Stormorken syndrome. To enable tunable inhibition, we further design oligomeric, optogenetic (Opto-CRAB), and chemogenetic (Chemo-CRAB) variants, providing graded and real-time control of CRAC activity. Chemo-CRAB further suppresses Ca2+ signaling downstream of RTKs, GPCRs, and CAR-T cell activation, establishing broad applicability across physiological and synthetic contexts. Together, these programmable peptide-based inhibitors provide a versatile platform to dissect SOCE dynamics and hold promise as a therapeutic strategy against autoimmune, inflammatory, and neoplastic disorders driven by CRAC channel hyperactivity. Store-operated Ca2+ entry is essential for cellular signalling, yet excessive calcium influx drives disease. Here, authors develop genetically encoded CRAC channel inhibitory binders (CRABs) to precisely modulate Ca2+ signalling, with therapeutic potential in channelopathies and cancer immunotherapy.