Abstract
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 short hairpin RNA-based gene-silencing assays. This screen utilized the most extensive genome-wide ORF collection to date, covering 90% of human, nonredundant, protein-encoding genes. We identified two ABCG1 (ABC subfamily G member 1) variants, which regulate cellular cholesterol, as modulators of hypotonicity-induced ATP release. We found that cholesterol levels control volume-regulated anion channel-dependent ATP release. These findings reveal novel mechanisms for the regulation of ATP release and volume-regulated anion channel activity and provide critical links among cellular status, cholesterol, and purinergic signaling.
Highlights
Purinergic signaling by extracellular ATP regulates a variety of cellular events and is implicated in both normal physiology and pathophysiology
2 The abbreviations used are: P2Y receptors (P2YRs), P2Y receptor; ANOVA, analysis of variance; volumeregulated anion channel (VRAC), volume-regulated anion channel; shRNA, short hairpin RNA; LOF, loss-of-function; GOF, gain-of-function; ABC, ATP-binding cassette; muscarinic-type acetylcholine receptors (mAChRs), muscarinic-type acetylcholine receptor; MCD, methyl--cyclodextrin; CALHM, calcium homeostasis modulator; PLC, phospholipase C; of ATP compared with intracellular levels (ϳ1–10 nM versus 1–10 mM, respectively) [2,3,4,5,6,7]
Our studies further demonstrate that ABCG1 modulates hypotonicity-induced ATP release through LRRC8Acontaining VRACs in a cholesterol-dependent manner
Summary
Hypotonicity induces ATP release [5, 6], which we observed by performing a luciferin–luciferase bioluminescence assay with cerebellar granule neurons treated for 30 s with a hypotonic solution (final concentration, 250 mmol/kg) (Fig. 1A). Hypotonicity-induced calcium responses and ATP release were similar between treatments with vehicle and 100 M ARL67156 to block extracellular nuclease activity in both ABCG1- and RFP-transfected HEK cells (Fig. S1) From these results, we concluded that ABCG1 increases ATP release upon hypotonic stimulation. We quantified ATP release from these HEK cells 30 s after hypotonic stimulation by using the luciferin–luciferase bioluminescence assay and found that cholesterol depletion via MCD treatment enhanced the amount of ATP released into the medium (Fig. 6D) This increased release was abolished by inhibiting VRAC activity with 20 M DCPIB (Fig. 6D). All of these results in neurons are consistent with what we observed in HEK cells, supporting the roles of cholesterol and VRAC in hypotonicity-induced ATP release
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