Mibefradil alters intracellular calcium concentration by activation of phospholipase C and IP₃ receptor function

Mibefradil is a tetralol derivative originally developed as an antagonist of T-type voltage-gated calcium (Ca²⁺) channels to treat hypertension when used at nanomolar dosage. More recently, its therapeutic application in hypertension has declined and has been instead repurposed as a treatment of cancer cell proliferation and solid tumor growth. Beyond its function as a Ca_v blocker, the micromolar concentration of mibefradil can stimulate a rise in [Ca²⁺]_cyt although the mechanism is poorly known. The chanzyme TRPM7 (transient receptor potential melastanin 7), the release of intracellular Ca²⁺ pools, and Ca²⁺ influx by ORAI channels have been associated with the increase in [Ca²⁺]_cyt triggered by mibefradil. This study aims to investigate the cellular targets and pathways associated with mibefradil's effect on [Ca²⁺]_cyt. To address these questions, we monitored changes in [Ca²⁺]_cyt in the specialized mouse epithelial cells (LS8 and ALC) and the widely used HEK-293 cells by stimulating these cells with mibefradil (0.1 μM to 100 μM). We show that mibefradil elicits an increase in [Ca²⁺]_cyt at concentrations above 10 μM (IC₅₀ around 50 μM) and a fast Ca²⁺ increase capacity at 100 μM. We found that inhibiting IP₃ receptors, depleting the ER-Ca²⁺ stores, or blocking phospholipase C (PLC), significantly decreased the capacity of mibefradil to elevate [Ca²⁺]_cyt. Moreover, the transient application of 100 μM mibefradil triggered Ca²⁺ influx by store-operated Ca²⁺ entry (SOCE) mediated by the ORAI channels. Our findings reveal that IP₃R and PLC are potential new targets of mibefradil offering novel insights into the effects of this drug.