Abstract <p>Efficient regulation of intracellular Ca²⁺ isessential for neuronal survival, and the sodium–calcium exchanger(NCX) is a major pathway for Ca²⁺ extrusion.NCX activity depends critically on the transmembrane Na⁺ gradientmaintained by the Na/K-ATPase (NKA). Although both proteins co-localize inneuronal lipid rafts, direct functional modulation of NCX by NKAhas not been quantitatively demonstrated. Here, we tested the hypothesisthat positive modulation of NKA enhances NCX forward-mode transportin cortical neurons. Using whole-cell patch-clamp electrophysiology,we recorded NCX current–voltage (I–V) relationships in rat cortical neuronsand applied 0.5 nM ouabain to selectively potentiate NKA. NCX currentswere isolated using KB-R7943, and experimentally obtained I–V curves werefitted with a well-established kinetic model to estimate intracellularNa⁺ concentration. Ouabain produced amarked rightward shift of the NCX I–V curve, increasing forward-modecurrent at physiological membrane potentials. Modeling indicateda reduction of intracellular Na⁺ from~32 mM to ~21 mM, consistent with NKA activation. In neurons loadedwith BAPTA, which inhibits NKA, ouabain no longer affected NCX parameters.These findings demonstrate that NKA-NCX interaction modulates NCXtransport by shaping the local Na⁺ gradient andprovide a mechanistic explanation for the neuroprotective actionsof low-nanomolar ouabain.</p>