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Much of the research on Na+/H+ exchange has been done in prokaryotic models, mainly on the NhaA Na+/H+-exchanger from Escherichia coli (EcNhaA). Two conserved aspartate residues, Asp-163 and Asp-164, are essential for transport and are candidates for possible binding sites for the two H+ that are exchanged for one Na+ to make the overall transport process electrogenic. More recently, a proposed mechanism of transport for EcNhaA has suggested direct binding of one of the transported H+ to the conserved Lys-300 residue, a salt bridge partner of Asp-163. This contention is supported by a study reporting that substitution of the equivalent residue, Lys-305, of a related Na+/H+ antiporter, NapA from Thermus thermophilus, renders the transporter electroneutral. In this work, we sought to establish whether the Lys-300 residue and its partner Asp-163 are essential for the electrogenicity of EcNhaA. To that end, we replaced Lys-300 with Gln, either alone or together with the simultaneous substitution of Asp-163 with Asn, and characterized these transporter variants in electrophysiological experiments combined with H+ transport measurements and stability analysis. We found that K300Q EcNhaA can still support electrogenic Na+/H+ antiport in EcNhaA, but has reduced thermal stability. A parallel electrophysiological investigation of the K305Q variant of TtNapA revealed that it is also electrogenic. Furthermore, replacement of both salt bridge partners in the ion-binding site of EcNhaA produced an electrogenic variant (D163N/K300Q). Our findings indicate that alternative mechanisms sustain EcNhaA activity in the absence of canonical ion-binding residues and that the conserved lysines confer structural stability.
In natural environments, background noise can degrade the integrity of acoustic signals, posing a problem for animals that rely on their vocalizations for communication and navigation. A simple behavioral strategy to combat acoustic interference would be to restrict call emissions to periods of low-amplitude or no noise. Using audio playback and computational tools for the automated detection of over 2.5 million vocalizations from groups of freely vocalizing bats, we show that bats (Carollia perspicillata) can dynamically adapt the timing of their calls to avoid acoustic jamming in both predictably and unpredictably patterned noise. This study demonstrates that bats spontaneously seek out temporal windows of opportunity for vocalizing in acoustically crowded environments, providing a mechanism for efficient echolocation and communication in cluttered acoustic landscapes.