Exploring the Li⁺ transporting mutant of NCX_Mj for assigning ion binding sites of mitochondrial NCLX

The plasma membrane (NCX) and mitochondrial (NCLX) Na⁺/Ca²⁺ exchangers are structurally related proteins, although they operate under strictly different ionic conditions and membrane potentials. In contrast with NCX, NCLX can transport either Li⁺ or Na⁺ in exchange for Ca²⁺. Whereas the crystal structure of the archaeal NCX (NCX_Mj) describes the binding sites for alternative binding of 3Na⁺ or 1Ca²⁺, these features remain elusive for NCLX due to the lack of structural information. To elucidate the ion-binding features of mitochondrial NCLX, we analyzed here the Li⁺-transporting NCLX_Mj mutant, produced by replacing the ion-coordinating residues in the archaeal NCX (NCX_Mj) to match the ion-coordinating residues of human NCLX. The NCLX_Mj-mediated Na⁺/Ca²⁺ or Li⁺/Ca²⁺ exchange rates are insensitive to varying voltage, consistent with an electroneutral ion exchange. Molecular dynamics (MD) simulations revealed that NCLX_Mj contains two novel Li⁺ binding sites with four ion-coordinating residues, derived from the three Na⁺ binding sites of NCX_Mj. The ion-coordination modes, observed in the MD simulations, were further supported by two-dimensional infrared (2D IR) spectroscopy and by testing the mutational effects on the ion fluxes. Collectively, our results revealed a structural basis for Li⁺ binding and electroneutral transport (2Na⁺/Li⁺:1Ca²⁺) by NCLX_Mj, meaning that the NCLX-mediated electroneutral transport may predefine mitochondrial Ca²⁺ and Na⁺ signaling to modulate cellular functions.