Poster V3 - Engineering of genetically encoded blockers for calcium channels

Introduction: The Ca2+ release-activated Ca2+ (CRAC) channel composed of stromal interaction molecule 1 (STIM1) and ORAI (ORAI1-3) constitutes a major calcium entry pathway in immune cells. STIM1 engages and gates ORAI calcium channels via its direct interaction with the C-termini of ORAI proteins, causing Ca2+ influx to trigger a cascade of cell signaling events. Mutations in STIM1 or ORAI1 are found in patients to cause immunodeficiency. Genetic mutations in STIM1 or ORAI1 lead to immunodeficiency, while the hyperactivation of CRAC channels due to the STIM1-R304W mutation results in Stormorken syndrome, a rare autosomal dominant disorder characterized by a constellation of symptoms including thrombocytopenia, miosis, mild bleeding diathesis, asplenia, intellectual disability, and tubular aggregate myopathy. Consequently, there is active exploration of chemical or peptide modulators targeting the CRAC channel as potential therapeutics for diseases associated with dysregulated CRAC channel activity in humans.

Materials and

Methods: Key methods used in this study include high-resolution confocal imaging for colocalization and puncta visualization, NFAT nuclear translocation assay, NFAT-dependent luciferase reporter assay, calcium influx assay, and electrophysiological measurements of CRAC channel current. A zebrafish embryo model of Stormorken syndrome was used to assess the therapeutic effect of engineered peptides.

Results, Conclusions, and Discussions: CRAC channel blocker (CRAB) peptides controlled by light (Opto-CRAB) and chemical (Chemo-CRAB) were introduced herein to treat diseases associated with gain-of-function mutations in ORAI1 and STIM1, for instance, tubular aggregate myopathy (TAM) and Stormorken syndrome. We show that a 20-mer peptide derived from ORAI C-terminal tail (ORAI-CT) fused with an oligomerization tag strongly associates with STIM1, thereby competitively blocking CRAC channel mediated Ca2+ signals and its downstream effects. In addition, we integrated optical or chemical dimerization systems with ORAI-CT to develop Opto-CRAB and Chemo-CRAB, respectively, to reversibly control the subcellular localization of ORAI-CT light or chemical, subsequently antagonizing the CRAC channel activity. Opto-CRAB and Chemo-CRAB enable reversible control over ORAI-CT subcellular localization and calcium-dependent signaling, thereby offering promising precise strategies for modulating CRAC channel activity, as demonstrated by optogenetic inhibition of T cell activation and amelioration of thrombocytopenia in a zebrafish model of Stormorken syndrome.

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