Alkalization-induced Ca²⁺-release from LBs is P2X4 dependent. (A) ATII cells labeled with fura 2-AM and expressing WT P2X4 tagged with mCherry (P2X₄(WT)-mCherry). P2X₄ is predominantly localized on LBs (arrowheads). Bar, 10 µm. (B) Top left: Image depicts the boxed area in A and highlights the perivesicular region (area between circles) around a P2X₄(WT)-mCherry–positive LB where changes in the fura-2 ratio were recorded. Image series illustrates changes in fura-2 ratio after addition of 10 mM NH₄Cl (at t = 40 s) in the same area as the image on the left. Bar, 5 µm. Bottom left: Normalized fura-2 ratios measured within a 1-µm perivesicular region around individual LBs (as illustrated in A, top) in control cells (blue) and cells (over)expressing P2X₄(WT)-mCherry (red) treated with 10 mM NH₄. Data represent means from 14 and 11 cells, respectively. Right: Quantification of the NH₄-induced fura-2 peak amplitude. Overexpression of P2X₄(WT)-mCherry, significantly increased amplitude (**, P = 0.0003).