Mechanism of action of two potent LRRC8/VRAC channel inhibitors

Volume-Regulated Anion Channels (VRACs), composed of Leucine-Rich Repeat Containing 8 (LRRC8) proteins, are emerging as promising therapeutic targets, but their pharmacology is poorly defined. Small-molecule VRAC inhibitors share lipophilic properties and exhibit a wide range of off-target effects, rendering them unsuitable for physiological studies. Furthermore, the mechanisms of action underlying their on- and off-target effects remain largely unclear. Here, we show that two structurally unrelated small-molecule inhibitors of VRACs, DCPIB and dicumarol, exert their cellular effects by accumulating in and permeating the cell membrane in an albumin-dependent and VRAC-independent manner. In conditions lacking serum/albumin, both compounds not only inhibit VRAC function but also disrupt store-operated Ca²⁺ entry (SOCE), Ca²⁺ signaling, and the activation and function of human and mouse T cells. Mechanistically, we show that DCPIB and dicumarol depolarize the mitochondrial membrane potential, leading to disruption of Ca²⁺ signaling, increased oxidative stress, actin aggregation, and apoptosis in T cells. These adverse effects are completely mitigated by the presence of serum/albumin in the buffer or culture media. Interestingly, while deleting LRRC8A and LRRC8C protects T cells from VRAC-mediated cell death, DCPIB and dicumarol failed to mimic this effect under standard culture conditions, suggesting that the ability of these VRAC inhibitors to accumulate or permeate the cell membrane is critical for inhibiting LRRC8/VRAC transport. Our results demonstrate that even though DCPIB and dicumarol are potent inhibitors of VRACs, their use in functional studies may be limited by their cell permeability and off-target effects on Ca²⁺ signaling.