Internalization of membrane and NCX1 exchangers during Ca-promoted MEND with activation by ATP perfusion. (A) Uptake of FM 4–64 dye (8 µM) in a BHK cell subjected to a Ca transient in the absence of ATP followed by cytoplasmic perfusion of 2 mM ATP. As indicated below the Cm record, the cell was maintained in standard solutions without ATP or GTP for 3 min. Outward NCX1 current was activated with 2 mM of extracellular Ca for 10 s, and ∼2 min later, 2 mM ATP and 0.2 mM GTP were perfused into the cell. During the experiment, FM dye was applied and removed multiple times to monitor dye binding to the outer cell surface versus dye that had been internalized. After the Ca-activated exocytic response, surface fluorescence is increased 34% more than expected from the increase of Cm. Thereafter, the introduction of nucleotides causes a 65% decrease of Cm, and the “washable” fluorescence decreases by 60% with a corresponding increase of “unwashable” fluorescence. Dye that is trapped in vesicles and vacuoles forms a clear rim close to the cell surface (see inset and Video 1; calibration bar, 10 µm). (B) Internalization of NCX1–pHluorin fusion protein during MEND in a T-REx-293 cell. Using the same ATP perfusion protocol as in A, fluorescence originating from NCX1 at the cell surface was defined by rapid pH jumps from 6 to 8. Subsequent to the decline of Cm by ∼45%, the NCX1 fluorescence at the cell surface is decreased by >30%. This internalized pH-insensitive florescence corresponds to the percentage of NCX1 exchangers internalized during MEND, as internalized membrane does not enter acidified compartments.