["The Journal of Physiology, EarlyView. ", "\nAbstract figure legend Due to its presence inside the dyadic cleft, Na+/Ca2+ exchanger (NCX) builds a functional unit together with L‐type calcium channels and ryanodine receptors in the dyadic cleft. NCX acts bidirectionally (forward and reverse mode) dependent on extracellular calcium ([Ca2+]o) and sodium ([Na+]o) concentrations and the membrane potential (phase of the cardiac cycle). At low [Ca2+]o and high [Na+]o, NCX acts in forward mode at rest, helping thereby to maintain cytosolic Ca2+ ([Ca2+]i) low. Inhibiting NCX causes an increase in spark frequency under these conditions as triggering [Ca2+]i increases. If [Ca2+]o is increased and/or [Na+]o decreased on the other hand, NCX acts in reverse mode at rest, increasing spark frequency that can then be reduced again by inhibiting NCX. During the early phase of action potential, when [Na+]i is already increased, NCX acts in reverse mode independent of the tested extracellular ion concentrations. This accelerates transsarcolemmal Ca2+ influx, as early [Ca2+]i increase is delayed with NCX inhibition. When [Ca2+]i rises during the cardiac cycle, NCX shifts to forward mode and supports Ca2+ extrusion. Overall, inhibiting NCX leads over time to an increase in diastolic [Ca2+]i. Created in BioRender. Hüttemeister, J. (2025).\n\n\n\n\n\n\n\n\n\nAbstract\nThe Na+/Ca2+ exchanger (NCX) transports Ca2+ and Na+ through the plasma membrane of cardiomyocytes. NCX dysregulation has been related to diastolic dysfunction. NCX inhibition has been identified as a potential therapeutic approach. It can accelerate the decay of the cytosolic Ca2+ concentration ([Ca2+]i) and improve impaired cardiomyocyte relaxation. We hypothesized that this counterintuitive effect is explained by the subcellular arrangement of NCX and local ion gradients within the intracellular Ca2+ release units. In a parallel model‐based and experimental approach, we re‐evaluated the location of NCX with regard to the dyadic cleft and its role in modulating [Ca2+]i. Stimulated emission depletion imaging revealed NCX in close proximity to junctophilin (the marker for the dyadic cleft). We simulated [Ca2+] dynamics in the dyadic cleft considering Ca2+ channels, NCX molecules and local concentration gradients. Positioning NCX inside the dyadic cleft in our computational model matched its action on spark rate. In forward mode (Ca2+ out, Na+ in) NCX decreased spontaneous Ca2+ release events (spark rate) in simulations and imaging experiments, while in reverse mode it increased them. In paced cardiomyocytes, NCX inhibition consistently increased diastolic [Ca2+]. The effects of NCX inhibition on transient amplitude and peak, however, depended on extracellular [Ca2+]o suggesting a role of reverse‐mode NCX activity at high [Ca2+]o. NCX inhibition prolonged the early rise of [Ca2+], corroborating that reverse‐mode NCX facilitates the rapid initial increase of [Ca2+]i during excitation. Our combined imaging, modelling and functional data support the hypothesis that NCX resides in the dyadic cleft where it bidirectionally shapes Ca2+ transients and spark activity.\n\n\n\n\n\n\n\n\n\nKey points\n\nOur data suggest that Na+/Ca2+ exchanger (NCX) is localized inside the dyadic cleft in cardiomyocytes, close to junctophilin, as shown by stimulated emission depletion imaging.\nComputational modelling confirmed NCX's dyadic positioning. It is critical for its effects on local Ca2+ signalling.\nNCX bidirectionally modulates Ca2+ dynamics: forward mode reduces, reverse mode increases spontaneous Ca2+ sparks.\nNCX inhibition raises diastolic cytosolic Ca2+ and alters Ca2+ transient amplitude depending on extracellular Ca2+ levels.\nReverse‐mode NCX facilitates rapid initial Ca2+ rise during excitation, highlighting its role in cardiac Ca2+ regulation and potential therapy.\n\n\n"]