pH-dependent activation of the Na

Na⁺/H⁺ antiporters are vital for regulating intracellular pH and sodium ion levels across all domains of life. In Escherichia coli, NhaA is the principal Na⁺/H⁺ antiporter, exhibiting strong pH sensitivity and rapid turnover, yet the structural transitions underlying its activation and substrate recognition have remained obscure. Here, we use single-particle cryo-electron microscopy to determine the conformational ensemble of NhaA across a physiological pH range and in the presence of Na⁺, complemented by constant-pH molecular dynamics simulations. High-resolution structures of apo and Na⁺-bound NhaA reconstituted in lipid nanodiscs reveal progressive opening of the cytoplasmic funnel with increasing pH. We also visualize the previously unresolved N-terminal tail, which forms a dynamic plug at the cytoplasmic entrance under low-pH conditions and disengages at alkaline pH, coinciding with activation. The Na⁺-bound structure captures Na⁺ coordination at the ion-binding site, and simulations suggest potential roles for the conserved charged residues. Together, these findings illuminate how pH sensing, N-terminal gating, and substrate binding are structurally coordinated in NhaA, providing a framework for understanding Na⁺/H⁺ antiporter activation and regulation, and the basis for targeting clinical important antiporters.