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The plasma membrane (NCX) and mitochondrial (NCLX) Na(+)/Ca(2+) 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 Ca(2+). Whereas the crystal structure of the archaeal NCX (NCX_Mj) describes the binding sites for alternative binding of 3Na(+) or 1Ca(2+), 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(+)/Ca(2+) or Li(+)/Ca(2+) 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 2D infrared 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(+):1Ca(2+)) by NCLX_Mj, meaning that the NCLX-mediated electroneutral transport may predefine mitochondrial Ca(2+) and Na(+) signaling to modulate cellular functions. |
