Trifluoperazine Elevates Intracellular Ca2+ Levels and Locks Open the Store‐Operated Calcium Entry Channels in Astrocytes

ABSTRACT Trifluoperazine (TFP), a known inhibitor of Ca 2+ ‐bound calmodulin (Ca 2+ /CaM), has been reported to elevate cytosolic Ca 2+ levels by disinhibiting inositol 1,4,5‐triphosphate receptor 2 (IP 3 R2), thereby suppressing glioblastoma invasion and inducing apoptosis. Interestingly, TFP induces a sustained Ca 2+ plateau, sensitive to extracellular Ca 2+ , suggesting involvement of Ca 2+ entry such as store‐operated calcium entry (SOCE). However, the underlying molecular mechanism remains elusive. Here, we report that TFP induces sustained Ca 2+ signals by blocking the Ca 2+ /CaM‐dependent desensitization of SOCE channels in cortical astrocyte cultures. TFP induces a prolonged Ca 2+ response, with distinct kinetics compared to other Ca 2+ modulators such as TFLLR‐NH 2 (a G αq ‐coupled GPCR agonist) and thapsigargin (a sacro/endoplasmic reticulum Ca 2+ ‐ATPase inhibitor). Under extracellular Ca 2+ ‐free conditions, Ca 2+ levels increase without reaching a plateau, suggesting that the sustained Ca 2+ signal relies on Ca 2+ influx. Pharmacological analysis shows that sustained Ca 2+ signals by TFP are CaM‐dependent. Gene silencing targeting STIM1 and Orai1–3 confirmed their essential roles in the sustained response. We find that TFP effectively “locks open” SOCE channels by inhibiting their desensitization, maintaining SOCE activity. This effect is also observed in ex vivo hippocampal dentate gyrus astrocytes. Structural modeling supports a mechanism in which TFP disrupts the interaction between Ca 2+ /CaM and the SOAR domain of STIM1. Together, these findings indicate that TFP elevates cytosolic Ca 2+ levels by maintaining SOCE activation, offering novel insights into the molecular actions of this drug. TFP can be a pharmacological tool for SOCE research as it locks SOCE channels open.