Exploring Structural and Functional Aspects of the CRAC Channel in Ascaris lumbricoides

Calcium (Ca²⁺) entry into cells, mediated by the CRAC channel (Calcium Release-Activated Calcium Channel), is essential for regulating various cellular processes. This pathway involves the coupling of STIM proteins, located in the endoplasmic reticulum (ER), with Orai channels in the plasma membrane (PM), which are highly selective for Ca²⁺. While well characterized in humans, this mechanism remains poorly understood in parasitic helminths that affect over one billion people worldwide, particularly in areas with poor sanitation. In this study, we investigated the activation mechanisms of CRAC channels in Ascaris lumbricoides (Al), a species for which no previous characterization of these channels has been reported. We observed that the protein EGFP-AlSTIM is not able to form puncta structures after Ca²⁺ depletion unless mCherry-AlOrai is co-expressed in HEK293 cells, unlike human STIM, which can form puncta independently of Orai. This phenomenon has been attributed to the absence of a polybasic domain (PBD) at the C-terminal end of AlSTIM, which is essential for anchoring to the plasma membrane. Conservation analyses revealed fewer basic residues (Lys/Arg) in the SOAR domain of A. lumbricoides, potentially impairing lipid interactions. Using NFAT-GFP nuclear translocation as a functional sensor of Ca²⁺ influx, we confirmed that the CRAC channel from A. lumbricoides is functional only when both EGFP-AlSTIM and mCherry-AlOrai are co-expressed. Furthermore, AlSTIM was not able to activate Orai from H. sapiens or S. mansoni, suggesting species-specific STIM-Orai pairing requirements. Additionally, the channel exhibited sensitivity to intracellular alkalinization (pH 9.0), indicating that EGFP-AlSTIM also acts as a pH sensor. This effect is likely mediated by conformational changes due to histidine residue deprotonation, which can serve as a molecular trigger for STIM activation. Our results highlight the structural and functional specificity of the CRAC channel components in helminths and provide a potential basis for the rational design of drugs targeting these organisms with high pharmacological selectivity.