Bcl-2 upregulates calcium efflux through PMCA and NCX1 to preserve intracellular calcium homeostasis and confer resistance to apoptosis

• Bcl-2 simultaneously enhances Ca²⁺ influx and efflux to maintain intracellular Ca²⁺ homeostasis. • Bcl-2 induces spontaneous Ca²⁺ oscillations by upregulating SOCE components. • Bcl-2 promotes Ca²⁺ efflux by increasing PMCA and NCX1 expression. • Inhibition of PMCA or NCX1 suppresses oscillations and causes cytosolic Ca²⁺ accumulation. • Bcl-2-mediated Ca²⁺ regulation contributes directly to its anti-apoptotic function. Bcl-2 has been shown to regulate intracellular calcium (Ca²⁺) homeostasis, thereby influencing cellular metabolism, survival, and apoptosis. While previous studies have primarily focused on Bcl-2’s role in modulating Ca²⁺ levels within the endoplasmic reticulum and mitochondria, extracellular Ca²⁺ is a major determinant of cellular activation and Ca²⁺ homeostasis. Therefore, it is important to investigate whether Bcl-2 also contributes to the regulation of Ca²⁺ flux across the plasma membrane. A vector control, wild-type Bcl-2, and a Bcl-2 mutant were stably expressed in MDCK (Madin-Darby Canine Kidney) cells that lacked endogenous Bcl-2 expression. Real-time intracellular Ca²⁺ measurements by a single-cell fluorimeter were performed to measure Ca²⁺ release and influx with the ratiometric Ca 2+ indicator Fura-2 AM. Our results demonstrate that Bcl-2 enhances store-operated Ca²⁺ entry (SOCE)-mediated Ca²⁺ influx, a key mechanism underlying spontaneous Ca²⁺ oscillations. Furthermore, Bcl-2 upregulates the expression of plasma membrane Ca²⁺ ATPase (PMCA) and Na + -Ca²⁺ exchanger 1 (NCX1), which mediate Ca²⁺ extrusion from the cytosol. Pharmacological inhibition of PMCA with resveratrol (RES) and of NCX1 with ORM-10103 suppressed spontaneous Ca²⁺ oscillations, with PMCA playing a more dominant role than NCX1. Additionally, both RES and ORM-10103 exacerbated thapsigargin-induced Ca²⁺ cytotoxicity. Collectively, our findings reveal that Bcl-2 promotes Ca²⁺ influx to sustain oscillatory signaling and facilitates Ca²⁺ efflux to prevent cytotoxic accumulation, thereby maintaining Ca²⁺ homeostasis. This dual regulatory function provides new insights into Bcl-2–mediated Ca²⁺ signaling and its biological significance in apoptosis inhibition, independent of its interactions with other Bcl-2 family proteins.