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

Trifluoperazine (TFP), a known inhibitor of Ca2+-bound calmodulin (Ca2+/CaM), has been reported to elevate cytosolic Ca2+ levels by disinhibiting inositol 1,4,5-triphosphate receptor 2 (IP3R2), thereby suppressing glioblastoma invasion and inducing apoptosis. Interestingly, TFP induces a sustained Ca2+ plateau, sensitive to extracellular Ca2+, suggesting involvement of Ca2+ entry such as store-operated calcium entry (SOCE). However, the underlying molecular mechanism remains elusive. Here, we report that TFP induces sustained Ca2+ signals by blocking the Ca2+/CaM-dependent desensitization of SOCE channels in cortical astrocyte cultures. TFP induces a prolonged Ca2+ response, with distinct kinetics compared to other Ca2+ modulators such as TFLLR-NH2 (a G alpha q-coupled GPCR agonist) and thapsigargin (a sacro/endoplasmic reticulum Ca2+-ATPase inhibitor). Under extracellular Ca2+-free conditions, Ca2+ levels increase without reaching a plateau, suggesting that the sustained Ca2+ signal relies on Ca2+ influx. Pharmacological analysis shows that sustained Ca2+ 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 Ca2+/CaM and the SOAR domain of STIM1. Together, these findings indicate that TFP elevates cytosolic Ca2+ 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.