Species-Specific Coupling, Pharmacological Targeting and Calmodulin Regulation of STIM–Orai CRAC Channel Activation in Parasitic Helminths

Store-operated Ca²⁺ entry (SOCE), mediated by the STIM–Orai complex (CRAC channel), is a central signalling pathway in metazoans that remains poorly explored in parasitic helminths. We integrated functional, in silico and biochemical approaches to investigate CRAC channel regulation in Ascaris lumbricoides (Al) and Schistosoma mansoni (Sm). In HEK293 cells, EGFP-AlSTIM remained diffuse following thapsigargin-induced ER Ca²⁺ depletion and formed puncta at ER–PM junctions only when co-expressed with its cognate Orai (mCherry-AlOrai), a behavior potentially related to the absence of the C-terminal polybasic domain in AlSTIM. GFP-NFAT nuclear translocation confirmed that the AlSTIM–AlOrai pair constitutes a functional CRAC channel, whereas the isolated C-terminal region of AlSTIM (containing SOAR) activated AlOrai constitutively. Cross-species experiments revealed asymmetric coupling specificity: AlSTIM activated neither human nor SmOrai, whereas human STIM1 efficiently activated AlOrai, indicating that selectivity resides mainly within STIM. Intracellular pH clamping with nigericin revealed that AlSTIM acts as a pH sensor with an activation window shifted towards alkaline values, consistent with histidines located in flexible loops rather than in the α-helical SOAR. Alignments and structural modelling showed conservation of the AlOrai transmembrane helices. The homo-hexameric AlOrai model (template PDB 7KR5) revealed two potentially druggable pore cavities (drug scores 0.66 and 0.45); virtual screening of 3,400 approved or clinical-stage drugs, followed by focused redocking, identified three top-ranked candidates (−12.2 to −11.1 kcal/mol): a pyrrolidinyl-4-pyrimidinyl steroidal derivative, golvatinib and rebastinib. During 50ns molecular dynamics in a lipid bilayer, all three remained stable within the pore, with persistent hydrophobic and π–alkyl contacts and conserved anchoring by Arg8 and Leu12, supporting the AlOrai pore as a potential site for pharmacological targeting. Given the reported involvement of calmodulin in CRAC channel inactivation in humans, we sought to investigate whether a similar regulatory mechanism is conserved in helminths. S. mansoni calmodulin was cloned into pET-28a(+), confirmed by sequencing and expressed as a soluble recombinant protein of 16.8 kDa, currently being purified for interaction assays with SOAR/STIM–Orai. Taken together, these results define the helminth CRAC channel as structurally conserved yet species-specifically regulated, and highlight the Orai pore as a promising target for new antiparasitic strategies.