ABC transporters control ATP release through cholesterol-dependent volume-regulated anion channel activity

Purinergic signaling by extracellular ATP regulates a variety of cellular events and is implicated in both normal physiology and pathophysiology. Several molecules have been associated with the release of ATP and other small molecules, but their precise contributions have been difficult to assess because of their complexity and heterogeneity. Here, we report on the results of a gain-of-function screen for modulators of hypotonicity-induced ATP release using HEK-293 cells and murine cerebellar granule neurons, along with bioluminescence, calcium FLIPR, and shRNA-based gene silencing assays. This screen utilized the most extensive genome-wide open reading frame collection to date, covering 90% of human, non-redundant, protein-encoding genes. We identified two ATP-binding cassette subfamily G member 1 (ABCG1) variants, which regulate cellular cholesterol, as modulators of hypotonicity induced ATP-release. We found that cholesterol levels control volume-regulated anion channel (VRAC)-dependent ATP release. These findings reveal novel mechanisms for the regulation of ATP release and VRAC activity and provide critical links among cellular status, cholesterol, and purinergic signaling.