Functional asymmetry of bidirectional Ca2+-movements in an archaeal sodium–calcium exchanger (NCX_Mj)

• The turnover rates of the ion-exchange cycle are 103–104 times slower in NCX_Mj than in mammalian NCX.
• Asymmetric Ca2+ movements represent a basic mechanism that diversifies Km values at opposite sides.
• Eukaryotic and prokaryotic NCX proteins share a common mechanism for asymmetric Ca2+ movements.
• Regulatory domains of mammalian NCX secondarily modulate the asymmetric Ca2+ movements.

Dynamic features of Ca2+ interactions with transport and regulatory sites control the Ca2+-fluxes in mammalian Na+/Ca2+(NCX) exchangers bearing the Ca2+-binding regulatory domains on the cytosolic 5L6 loop. The crystal structure of Methanococcus jannaschii NCX (NCX_Mj) may serve as a template for studying ion-transport mechanisms since NCX_Mj does not contain the regulatory domains. The turnover rate of Na+/Ca2+ exchange (kcat = 0.5 ± 0.2s−1) in WT–NCX_Mj is 103–104 times slower than in mammalian NCX. In NCX_Mj, the intrinsic equilibrium (Kint) for bidirectional Ca2+ movements (defined as the ratio between the cytosolic and extracellular Km of Ca2+/Ca2+ exchange) is asymmetric, Kint = 0.15 ± 0.5. Therefore, the Ca2+ movement from the cytosol to the extracellular side is ∼7-times faster than in the opposite direction, thereby representing a stabilization of outward-facing (extracellular) access. This intrinsic asymmetry accounts for observed differences in the cytosolic and extracellulr Km values having a physiological relevance. Bidirectional Ca2+ movements are also asymmetric in mammalian NCX. Thus, the stabilization of the outward-facing access along the transport cycle is a common feature among NCX orthologs despite huge differences in the ion-transport kinetics. Elongation of the cytosolic 5L6 loop in NCX_Mj by 8 or 14 residues accelerates the ion transport rates (kcat) ∼10 fold, while increasing the Kint values 100–250-fold (Kint = 15–35). Therefore, 5L6 controls both the intrinsic equilibrium and rates of bidirectional Ca2+ movements in NCX proteins. Some additional structural elements may shape the kinetic variances among phylogenetically distant NCX variants, although the intrinsic asymmetry (Kint) of bidirectional Ca2+ movements seems to be comparable among evolutionary diverged NCX variants.

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