ANO5-dependent Ca²⁺-PLS is associated with an ionic current. (a) Simultaneous measurement of annexin V–Alexa Fluor 568 binding and whole-cell patch clamp recording of ANO5-Clover HEK293 cells. Cells were patch-clamped with 200 µM free Ca²⁺ in the intracellular (pipette) solution, and currents were measured from a holding potential of 0 mV by voltage steps between −100 mV and +100 mV in 20-mV increments. Representative images (top) and corresponding currents (bottom) show a typical experiment of 29. The first panel is a bright-field image of the cell, and the second two images show fluorescence of bound annexin V. Scale bar, 10 μm. (b) Number of cells scrambled during patch-clamp experiments. White numbers indicate the number of cells. (c) Mean I–V relationships before (black squares) and after (red circles) cells showed significant annexin V binding. (d) Quantification of the percentage of cells bound by annexin V–Alexa Fluor 568 in ANO5-Clover or ANO6-Clover–expressing HEK293 cells when stimulated via whole-cell patch clamp as in panel a but with different concentrations of free Ca²⁺ in the intracellular (pipette) solution. Each condition represents data from >10 cells.