Authors:

Richard Song

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About Paper:

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 [1]. Furthermore, the mechanisms of action underlying their on- and off-target effects remain largely unclear. Here, we use intracellular calcium measurements, cell viability assays, and membrane permeability assays to show that the best-in-class small-molecule inhibitor of VRACs, DCPIB, exerts its cellular effects by accumulating in and permeating the cell membrane in an albumin-dependent and VRAC-independent manner. Additionally, we find that in conditions lacking serum or albumin, DCPIB not only inhibits VRAC function but also disrupts store-operated Ca** entry (SOCE), Ca?* signaling, and the activation and function of human and mouse T cells. Mechanistically, we use mitochondrial function assays, fluorescence microscopy, and western blotting to show that DCPIB depolarizes the mitochondrial membrane potential, leading to disruption of Ca** signaling, increased oxidative stress, actin aggregation, and apoptosis in T cells. These adverse effects are completely mitigated by the presence of serum or albumin in the buffer or culture media. Interestingly, while knocking out 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 DCPIB to accumulate or permeate the cell membrane is critical for inhibiting LRRC8/VRAC transport. Our results demonstrate that even though DCPIB is a potent inhibitor of VRACs, its use in functional studies may be limited by its cell permeability and off-target effects on Ca"* signaling. As such, we strongly recommend avoiding the use of DCPIB in functional in vitro and in vivo studies and suggest limiting its application to short-term electrophysiological experiments to prevent confounding effects.

Source:

University of Chicago

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Co-authors:

Richard Song