Transport mechanism and pH regulation of the Na⁺/H⁺ antiporter NhaA from Escherichia coli: An electrophysiological study

Using an electrophysiological assay the activity of NhaA was tested in a wide pH range from pH 5.0 to 9.5. Forward and reverse transport directions were investigated at zero membrane potential using preparations with inside-out and right side-out-oriented transporters with Na⁺ or H⁺ gradients as the driving force. Under symmetrical pH conditions with a Na ⁺ gradient for activation, both the wt and the pH-shifted G338S variant exhibit highly symmetrical transport activity with bell-shaped pH dependences, but the optimal pH was shifted 1.8 pH units to the acidic range in the variant. In both strains the pH dependence was associated with a systematic increase of the K_m for Na⁺ at acidic pH. Under symmetrical Na⁺ concentration with a pH gradient for NhaA activation, an unexpected novel characteristic of the antiporter was revealed; rather than being down-regulated, it remained active even at pH as low as 5. These data allowed a transport mechanism to advance based on competing Na⁺ and H⁺ binding to a common transport site and a kinetic model to develop quantitatively explaining the experimental results. In support of these results, both alkaline pH and Na⁺ induced the conformational change of NhaA associated with NhaA cation translocation as demonstrated here by trypsin digestion. Furthermore, Na⁺ translocation was found to be associated with the displacement of a negative charge. In conclusion, the electrophysiological assay allows the revelation of the mechanism of NhaA antiport and sheds new light on the concept of NhaA pH regulation.