Hypotonicity-induced purinergic signaling requires the ATPase activity of specific ATP transporters. Hypotonicity (final concentration, 250 mmol/kg)–induced calcium responses from HEK cells transfected transiently with ABC transporters, an ABCG1 mutant, or RFP (control) were measured with the calcium FLIPR assay. A, the ATPase activity of ABCG1 is required for hypotonicity-induced calcium responses. ABCG1, but not the ABCG1 mutant with a lysine-to-methionine substitution at position 124 (K124M) in the ABC, robustly increased hypotonicity-induced calcium responses (n = 4) (one-way ANOVA; F(2,9) = 501.7; p < 0.001). B, phylogenetic tree and hypotonicity-induced calcium responses of ABC transporters. Three ABC transporters (ABCG1, ABCG4, and ABCA1) among 39 ABC transporters increased the hypotonicity-induced calcium responses (n = 4) significantly relative to control (RFP). Eight ABC transporters were untested (N.A.). The dashed line indicates the response from RFP-transfected HEK cells (one-way ANOVA; F(39,120) = 35.04; p < 0.001). The data are means ± standard deviation (one-way ANOVA followed by Tukey's test (A and B)). *, p < 0.05; ***, p < 0.001.