(A) Cartoon showing the NCX transport modes activated by different external [Na+] and [Ca2+] conditions and their expected effects on the activities of ion pumps and the production of NADHCYT from glycolysis activation. Reverse NCX transport (left schematic) increases intracellular [Ca2+], which increases the activities Ca2+ pumps and Ca2+ transport into mitochondria (magenta arrows). Forward NCX transport (right schematic) increases intracellular [Na+], which increases the activity of the Na+/K+ pump (orange arrows). The bracket below each schematic indicates the NCX transport mode activated by the external solution changes in (C). Transport stoichiometries are not indicated. Abbreviations: Na+/Ca2+-exchanger (NCX), Na+/K+-ATPase (NKA), plasma membrane Ca2+-ATPase (PMCA), sarco-/endo-plasmic reticulum Ca2+-ATPase (SERCA), mitochondrial Ca2+ uniporter (MCU), mitochondrial Na+/Ca2+-exchanger (mNCX), endoplasmic reticulum (ER). (B) Representative fluorescence lifetime (LT) traces of Peredox (top trace) and RCaMP (bottom trace) from a DGC bathed in ACSF. Antidromic stimulation was delivered at the time point indicated by the arrow along the RCaMP trace, which transiently increases both NADHCYT and Ca2+CYT. (C) Fluorescence LT traces of Peredox (top) and RCaMP (bottom) from a DGC showing how external Na+ and Ca2+ changes affect NADHCYT and Ca2+CYT. The bars above the Peredox trace indicate the external [Na+] and [Ca2+]. NADHCYT was decreased by switching the bath solution from ACSF (147 mM Na+ and 2 mM Ca2+) to a solution with nominally 0 Na+ and 0 Ca2+ (cyan shading). Ca2+CYT was elevated by applying 0.5 mM Ca2+ with 0 Na+ to activate reverse NCX transport (magenta shading), and NADHCYT decreased further. NADHCYT was strongly increased after activating forward NCX transport by the subsequent removal of external Ca2+ and application of 147 mM Na+ (orange shading). (D) Box plots of the fluorescence LTs of Peredox (top) and RCaMP (bottom) showing the effects of the external Na+ and Ca2+ changes performed in panel C across many DGCs (n=53). The external bath conditions for each box plot are listed at the bottom of the RCaMP plot in chronological order from left to right. The colors of each box plot correspond to the colors indicated in (C). The mean Peredox LT values in each condition were: 1.63±0.06 ns in ACSF, 1.52±0.05 ns in 0 Na+ and 0 Ca2+, 1.46±0.09 ns in 0 Na and 0.5 mM Ca2+, and 1.72±0.06 ns in 147 mM Na+ and 0 Ca2+. The mean RCaMP LT values in each condition were: 0.88±0.05 ns in ACSF, 0.88±0.05 ns in 0 Na+ and 0 Ca2+, 1.52±0.15 ns in 0 Na+ and 0.5 mM Ca2+, and 0.93±0.07 ns in 147 mM Na+ and 0 Ca2+. (E) Changes to the Peredox LT relative to the 0 Na+ and 0 Ca2+ condition, after either a Ca2+CYT elevation from reverse NCX transport (Ca, black box, magenta filled diamonds), an influx of Na+ due to forward NCX transport (Na after Ca, black box, orange filled diamonds), or application of Na+ without forward NCX (Na no Ca, gray box, orange open diamonds). The mean Peredox LT changes were: –0.07±0.08 ns (n=53) for Ca2+CYT elevation, 0.20±0.05 ns (n=53) for Na+ influx via forward NCX, and 0.07364±0.04565 ns (n=19) for Na+ application without forward NCX. Statistical significance between ‘Ca’ and ‘Na after Ca’ is indicated by a paired Wilcoxon test and between ‘Na after Ca’ and ‘Na no Ca’ by a Mann-Whitney test. (F) Effect of external Na+ on the return of Ca2+CYT to baseline following a reverse NCX transport-mediated Ca2+ influx. The mean decay of the RCaMP LT following the Ca2+CYT increase (normalized to the peak RCaMP LT value) is shown when the external solution contained either 147 mM Na+ (solid line, orange SD shading, n=53) or 0 Na+ (dashed line, green SD shading, n=49). Decay data in 147 mM Na+ were from the same DGCs as in (D) and (E), while data in 0 Na+ were from the same DGCs as Figure 1—figure supplement 1B; a representative trace of this experiment is shown in Figure 1—figure supplement 1A.