Distinct Mechanisms of Sodium-Calcium Exchanger NCX1.4 Regulation by Ca²⁺, ATP, and PIP₂

Mammalian Na⁺/Ca²⁺ exchangers (NCXs) mediate inward (Ca²⁺-exit) or outward (Ca²⁺-entry) currents (I_NCX), with the peak current (I_peak) declining to the steady-state current (I_ss). The Ca²⁺-, ATP-, and PIP₂-dependent activation of cardiac (NCX1.1) inward I_NCX has been previously documented. To investigate possible interactions among regulatory modes, we examined the effects of ATP and PIP₂ on outward I_NCX when the CBD1 and CBD2 domains of NCX1.4 (brain variant) are Ca²⁺-occupied and -unoccupied. At nominally zero cytosolic Ca²⁺, ATP and PIP₂ significantly reduced the outward I_peak amplitude. While increasing cytosolic Ca²⁺ increased I_peak and I_ss/I_peak, consistent with CBD1- and CBD2-dependent regulation, no significant effects of ATP or PIP₂ were detected at 0.1-10 μM cytosolic Ca²⁺. These observations indicate distinct responses under the Ca²⁺ conditions examined and warrant direct mechanistic investigation. Although we observed opposing effects of ATP and PIP₂ on inward (in NCX1.1) and outward (in NCX1.4) currents using different patch-clamp configurations and protocols, the data collectively suggest that Ca²⁺, ATP, and PIP₂ regulatory modes may operate differently on the forward and reverse modes in NCX1.1 and NCX1.4 variants. Given the important physiological implications across distinct cell types (e.g., cardiomyocytes, neurons, glia, and kidney), careful investigation is needed to resolve how distinct regulatory modes affect the forward and reverse modes of mammalian NCX variants.