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

Abstract

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 GltPh and GltTk, 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 GltPh 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 GltTk 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.

Evolutionary analysis reveals the origin of sodium coupling in glutamate transporters

“…Further computational studies will be needed to understand how the structured water molecules and side chain interactions stabilize the nearly perfectly formed Na + binding sites. Notably, mutating M311 in Na2 site of GT-Na to leucine found in GT-H does not significantly alter coupling, transport, or HP2 closure upon substrate binding (31,(33)(34)(35), consistent with the idea that uncoupling form the Na + ion in GT-H can occur through mechanisms other than disrupting the site.…”

“…In GT-Na transporters, Na + binding to Na1 and Na2 sites precedes substrate binding (19)(20)(21)(30)(31)(32). In the last step before transport domain translocation, Na2 'locks' HP2 closed over the bound substrate (28,31,(33)(34)(35). GT-H transporters have conserved substitutions in all three sites: T92[A/L/I/V/M], D405N, and M311L, respectively (Figure 1b-c).…”

“…Thus, coupled binding and transport occur because the high-affinity substrate binding state is a high-energy state only accessible when Na + binds. Accordingly, mutations in all three Na + -binding sites diminish coupled substrate binding and transport (3,6,(33)(34)(35)(36).…”

Structural snapshots of TRPV1 reveal mechanism of polymodal functionality

“…However, we observe Article a clear density that appears to be a cation coordinated by four sulfur atoms of M682 (Figures S6M and S6N). Although very rare, sulfur from methionine has been found to coordinate Na + (Guskov et al, 2016), with an atom-to-atom distance of 2.7 A (Zhou et al, 2021). If this density corresponds to a coordinated Na + , then the C 2 conformation is permeable to partially dehydrated cations.…”

Dancing with the ions: symport and antiport mechanisms of human glutamate transporters

“…In the absence of Na + ions, the motifs are in the “apo” configurations: the M367 sidechain points out of the substrate-binding site, and R447, salt-bridged to deprotonated proton carrier E347, occupies the site (Qiu et al ., 2021). Sodium binding to Na1 and Na3 sites induces the “bound” configuration: M367 swings into the binding pocket, poised to coordinate the third Na + ion in the Na2 site (Zhou et al, 2022). R447 disengages from E347 and swings out of the pocket, forming cation-ν interactions with Y373 and ready to coordinate the substrate.…”