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

Abstract

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 Km 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.

Species differences in bacterial NhaA Na+/H+ exchangers

“…This gave rise to negative transient currents of up to 5 nA in amplitude (Fig. 1B) with similar characteristics to those previously reported for EcNhaA [6,20] and StNhaA [12]. The negative polarity of these currents is in accordance to the overall transport of one negative charge per transport cycle to the interior of the liposomes.…”

“…1C and D), yielding the same value as for EcNhaA and a pK value which is shifted by less than one pH unit into the acidic. For comparison we also fitted data obtained previously for StNhaA to the kinetic model [12] and included the obtained kinetic parameters together with those from EcNhaA in Table 1.…”

“…The physiological role of EcNhaA is to enable growth of E. coli in high salinity media or in the presence of lithium [4]. Close homologues of EcNhaA have been found in other Enterobacteriaceae, including Vibrio cholerae [10], Salmonella enteritidis [11] , Salmonella typhimurium [12] and Helicobacter pylori [13]. We have recently characterized NhaA from Salmonella enterica serovar Typhimurium LT2 (StNhaA) using solid‐supported membrane (SSM)‐based electrophysiology and found a transport activity profile very similar to that of EcNhaA in respect to the transition from low to high pH [12], in line with the high sequence identity between the two (92%), though the transporter has a higher activity in the alkaline.…”

Aggregation/dispersion transitions of T4 phage triggered by environmental ion availability

“…Similar cation transporters have been found in other bacteria, including human pathogens [30]. Activity of these, mostly high-yield anti-porters, locally enrich the microenvironment with ions; thus a discrete ionic zone formed on the bacterial surface creates a distinct environment that differs from the surrounding soil or water.…”

A universal mechanism for transport and regulation of CPA sodium proton exchangers

“…Further electrophysiological investigation of a number of enterobacterial CPA2 NhaA Na + /H + exchangers yielded similar results as for EcNhaA: bell-shaped pH profiles at symmetrical pH and acidic down-regulation by substrate competition as predicted by the simple kinetic model (Calinescu et al, 2014a, Lentes et al, 2014. It should also be mentioned here that we could prove (Calinescu et al, 2014a) that the previously reported "pH-independent" behavior of the NhaA exchanger from Helicobacter pylori was just a result of the investigation method and the experimental conditions used.…”