Functional and optical analysis of the influence of MEND on NCX1. (A) MEND responses and NCX1 currents in a BHK cell perfused with a high (20 mM) EGTA concentration to negate Ca transients and high ATP (8 mM) to promote MEND reversal. For the most part, MEND reverses within 2 min. We note that this experiment was performed with standard solutions, not modified standard solutions that promote recovery. NCX1 current is decreased after each MEND response and recovers partially during recovery of Cm. (B) Time-lapse confocal imaging of an HEK293 cell stably expressing NCX1–pHluorin fusion protein before and after exposing the cell to 150 µM NP-40 for 10 s. Fluorescence arising from NCX1 at the cell surface is determined as the fluorescence activated by jumping extracellular pH from 6.0 to 8.0. MEND induced by NP-40 causes a Cm drop of 48%, whereas the fluorescence intensity of cell surface NCX1 (i.e., fluorescence jump on changing solutions from pH 6.0 to 8.0) decreases by 37%. The presence of fluorescence at pH 6.0 after MEND indicates that NCX1 has entered an internal compartment that does not immediately acidify.
