Interplay between the Conserved Pore Residues Thr-91 and His-209 Controls Formate Translocation through the FocA Channel

The formate channel A (FocA) belongs to the formate-nitrite transporter (FNT) family, members of which permeate small monovalent anions. FocA from Escherichia coli translocates formate/formic acid bi-directionally across the cytoplasmic membrane during fermentative growth. Two residues are particularly well-conserved within the translocation pores of FNTs: threonine-91 and histidine-209, based on E. coli FocA numbering. These residues are located at the tips of two broken transmembrane helices and control anion passage. H209 is the only charged residue within the pore and interacts with T91. Here, we addressed the role of the T91-H209 interaction network in the permeation of formate in vivo through FocA by performing an extensive amino acid-exchange study. Monitoring changes in intracellular formate using a formate-responsive fdhFP::lacZ reporter system revealed that T91 is essential for the ability of FocA to translocate formate bi-directionally. Only exchange for serine was partially tolerated, indicating that the hydroxyl group of T91 is mechanistically important. Substitution of H209 with N or Q was previously shown to convert FocA into a formate efflux channel. We show here that residue exchanges A, I, and T at this position resulted in a similar phenotype. Moreover, efflux function was confirmed for these FocA variants by measuring excreted formate in the culture medium. Substitution of bulky or charged residues for H209 prevented bi-directional formate passage. Studies using hypophosphite, a toxic analogue of formate taken up by FocA, and which causes impaired growth, confirmed that T91 and H209 substitutions essentially abolished, or drastically reduced, FocA’s translocation activity, as shown by effects on growth rate. The exceptions were T91S- and T91Y-exchange variants that retained partial ability to take up inhibitory hypophosphite. Together, our findings indicate that T91 is essential for formate permeation in both directions; however, it is particularly important to allow anion efflux. Moreover, H209 is essential for formate uptake by FocA, strongly suggesting that protonation-deprotonation of this residue plays a role in formate uptake. Finally, our results substantiate the premise that efflux and influx of formate by FocA are mechanistically distinct processes that are controlled by the interplay between T91 and H209.