On the Role of a Conserved Methionine in the Na(+)-Coupling Mechanism of a Neurotransmitter Transporter Homolog

Excitatory amino acid transporters (EAAT) play a key role in glutamatergic synaptic communication. Driven by transmembrane cation gradients, these transporters catalyze the reuptake of glutamate from the synaptic cleft once this neurotransmitter has been utilized for signaling. Two decades ago, pioneering studies in the Kanner lab identified a conserved methionine within the transmembrane domain as key for substrate turnover rate and specificity; later structural work, particularly for the prokaryotic homologs Glt(Ph) and Glt(Tk), revealed that this methionine is involved in the coordination of one of the three Na(+) ions that are co-transported with the substrate. Albeit extremely atypical, the existence of this interaction is consistent with biophysical analyses of Glt(Ph) showing that mutations of this methionine diminish the binding cooperativity between substrates and Na(+). It has been unclear, however, whether this intriguing methionine influences the thermodynamics of the transport reaction, i.e., its substrate:ion stoichiometry, or whether it simply fosters a specific kinetics in the binding reaction, which, while influential for the turnover rate, do not fundamentally explain the ion-coupling mechanism of this class of transporters. Here, studies of Glt(Tk) using experimental and computational methods independently arrive at the conclusion that the latter hypothesis is the most plausible, and lay the groundwork for future efforts to uncover the underlying mechanism. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s11064-021-03253-w) contains supplementary material, which is available to authorized users.