The three-dimensional crystal structure of Escherichia coli NhaA determined at pH 4 provided the first structural insights into the mechanism of antiport and pH regulation of a Na ؉ /H ؉ antiporter. However, because NhaA is activated at physiological pH (pH 6.5-8.5), many questions pertaining to the active state of NhaA have remained open including the structural and physiological roles of helix IX and its loop VIII-IX. Here we studied this NhaA segment (Glu 241 -Phe 267 ) by structure-based biochemical approaches at physiological pH. Cysteine-scanning mutagenesis identified new mutations affecting the pH dependence of NhaA, suggesting their contribution to the "pH sensor." Furthermore mutation F267C reduced the H ؉ /Na ؉ stoichiometry of the antiporter, and F267C/F344C inactivated the antiporter activity. Tests of accessibility to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide, a membrane-impermeant positively charged SH reagent with a width similar to the diameter of hydrated Na ؉ , suggested that at physiological pH the cytoplasmic cation funnel is more accessible than at acidic pH. Assaying intermolecular cross-linking in situ between single Cys replacement mutants uncovered the NhaA dimer interface at the cytoplasmic side of the membrane; between Leu 255 and the cytoplasm, many Cys replacements cross-link with various cross-linkers spanning different distances (10 -18 Å ) implying a flexible interface. L255C formed intermolecular S-S bonds, cross-linked only with a 5-Å cross-linker, and when chemically modified caused an alkaline shift of 1 pH unit in the pH dependence of NhaA and a 6-fold increase in the apparent K m for Na ؉ of the exchange activity suggesting a rigid point in the dimer interface critical for NhaA activity and pH regulation.Regulation of intracellular pH, cellular Na ϩ content, and cell volume is essential for all living cells. Na ϩ /H ϩ antiporters play primary roles in these crucial processes. They are integral membrane proteins, ubiquitous throughout the biological kingdom. Many Na ϩ /H ϩ antiporters are tightly regulated by pH, a property that underpins their capacity to maintain pH homeostasis of the cytoplasm (1).NhaA, the main Na ϩ /H ϩ antiporter of Escherichia coli, has eukaryotic orthologs, including human (2, 3). It is an electrogenic antiporter with a stoichiometry of 2 H ϩ /1 Na ϩ (1, 4) and is strongly dependent on pH; its rate of activity changes over 3 orders of magnitude between pH 7.0 and 8.5 (1, 4, 5).NhaA is a dimer in the native membrane as revealed by genetic complementation data, biochemical pulldown experiments, intermolecular cross-linking (6), ESR studies (7,8), and cryoelectron microscopy of two-dimensional crystals (9, 10). The recently determined crystal structure of NhaA monomer at pH 4 (11) has provided the first structural insights into the mechanism of antiport and pH regulation of a Na ϩ /H ϩ antiporter. NhaA consists of 12 TMSs 2 with the N and C termini pointing into the cytoplasm. It represents a novel fold; TMSs IV and XI are each comprised ...