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

Moshe Giladi, Sunayana Mitra, Luba Simhaev, Reuben Hiller, Bosmat Refaeli, Tali Strauss, Carlos R. Baiz, Daniel Khananshvili*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

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.

Original languageEnglish
Article number102651
JournalCell Calcium
Volume107
DOIs
StatePublished - Nov 2022

Funding

FundersFunder number
Kahn Foundation
Tel Aviv Medical Center
National Institutes of Health
National Institute of General Medical SciencesR35GM133359
National Institute of General Medical Sciences
Welch FoundationF-1891
Welch Foundation
Israel Cancer Research Fund19202
Israel Cancer Research Fund
Israel Science Foundation1351/18
Israel Science Foundation
Tel Aviv University

    Keywords

    • 2D IR
    • Ion selectivity
    • Ion-binding sites
    • NCLX
    • NCX
    • Sodium-calcium exchanger

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