Molecular Characterization of the Na(+)/H(+)-Antiporter NhaA from Salmonella Typhimurium

Na(+)/H(+) antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na(+)-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na(+)/H(+) antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na(+)/H(+) antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na(+)-sensitive phenotype of an E. coli strain that lacks the main Na(+)/H(+) antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na(+)(Li(+))/H(+)-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na(+)(Li(+))/2H(+) stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of K(m) (Na) (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K(+) does not affect Na(+) affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na(+)/H(+) antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies.