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

Abstract

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_αq-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.