Three two-component transporters with channel-like properties have monovalent cation/proton antiport activity

Properties of four two-component bacterial transport systems of the cation/proton antiporter-2 (CPA2) family led to suggestions that this CPA2 subset may use a channel rather than an antiport mechanism [see Booth IR, Edwards MD, Gunasekera B, Li C, Miller S (2005) in Bacterial Ion Channels, eds Kubalski A, Martinac B (Am Soc Microbiol, Washington, DC) pp 21-40]. The transporter subset includes the intensively studied glutathione-gated K+ efflux systems from Escherichia coli, KefGB, and KefFC. KefG and KefF are ancillary proteins. They are peripheral membrane proteins that are encoded in operons with the respective transporter proteins, Kef B and KefC, and are required for optimal efflux activity. The other two-component CPA2 transporters of the subset are AmhMT, an NH4+ (K+) efflux system from alkaliphilic Bacillus pseudofirmus OF4; and YhaTU, a K+ efflux system from Bacillus subtilis. Here a K+/H+ antiport capacity was demonstrated for YhaTU, AmhMT, and KefFC in membrane vesicles from antiporter-deficient E. coli KNabc. The apparent Km for K+ was in the low mM range. The peripheral protein was required for YhaU- and KefC-dependent antiport, whereas both AmhT and AmhMT exhibited antiport. KefFC had the broadest range of substrates, using Rb+approximate to K+> Li+> Na+. Glutathione significantly inhibited KefFC-mediated K+/H+ antiport in vesicles. The inhibition was enhanced by NADH, which presumably binds to the KTN/RCK domain of KefC. The antiport mechanism accounts for the H+ uptake involved in KefFC-mediated electrophile resistance in vivo. Because the physiological substrate of AmhMT in the alkaliphile is NH4+, the results also imply that AmhMT catalyzes NH4+/H+ antiport, which would prevent net cytoplasmic H+ loss during NH4+ efflux.