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

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 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 ⁺ :1Ca ²⁺ ) by NCLX_Mj, meaning that the NCLX-mediated electroneutral transport may predefine mitochondrial Ca ²⁺ and Na ⁺ signaling to modulate cellular functions.
Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest
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