Ano5 ^−/−muscle cells exhibit perturbed Ca²⁺-PLS and PLS-associated ionic current. (a) Simultaneous whole-cell patch clamp and annexin V–Alexa Fluor 568 binding of primary MPCs differentiated for ∼24 hr. Images on the left show bright-field micrographs of patch-clamped MPCs. Center and right images show annexin V–Alexa Fluor 568 binding. Scale bars, 10 μm. Below the images are ionic currents recorded using voltage steps between +100 and −100 mV in 20-mV increments from a holding potential of 0 mV. (b) Quantification of Ca²⁺-PLS in MPCs (left), and average current of differentiated myoblasts at +100 mV (right). The statistical difference in Ca²⁺-PLS between genotypes was calculated using a Fisher exact test (***, P = 0.0001). The current at 20 min was statistically evaluated using a paired Student's t test (*, P = 0.026). The white numbers in the columns indicate the number of cells assayed. The same cells were measured at 3 and 20 min. Error bars indicate SEM. (c) Western blot of native ANO6 levels in wild-type and Ano5 knockout MPCs differentiated for ∼24 h.