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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 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.

SUBMITTER: Zhou W

PROVIDER: S-EPMC8431971 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Neurotransmitter/Na(+) symporters (NSSs) terminate neuronal signalling by recapturing neurotransmitter released into the synapse in a co-transport (symport) mechanism driven by the Na(+) electrochemical gradient. NSSs for dopamine, noradrenaline and serotonin are targeted by the psychostimulants cocaine and amphetamine, as well as by antidepressants. The crystal structure of LeuT, a prokaryotic NSS homologue, revealed an occluded conformation in which a leucine (Leu) and two Na(+) are bound deep within the protein. This structure has been the basis for extensive structural and computational exploration of the functional mechanisms of proteins with a LeuT-like fold. Subsequently, an 'outward-open' conformation was determined in the presence of the inhibitor tryptophan, and the Na(+)-dependent formation of a dynamic outward-facing intermediate was identified using electron paramagnetic resonance spectroscopy. In addition, single-molecule fluorescence resonance energy transfer imaging has been used to reveal reversible transitions to an inward-open LeuT conformation, which involve the movement of transmembrane helix TM1a away from the transmembrane helical bundle. We investigated how substrate binding is coupled to structural transitions in LeuT during Na(+)-coupled transport. Here we report a process whereby substrate binding from the extracellular side of LeuT facilitates intracellular gate opening and substrate release at the intracellular face of the protein. In the presence of alanine, a substrate that is transported ?10-fold faster than leucine, we observed alanine-induced dynamics in the intracellular gate region of LeuT that directly correlate with transport efficiency. Collectively, our data reveal functionally relevant and previously hidden aspects of the NSS transport mechanism that emphasize the functional importance of a second substrate (S2) binding site within the extracellular vestibule. Substrate binding in this S2 site appears to act cooperatively with the primary substrate (S1) binding site to control intracellular gating more than 30?Å away, in a manner that allows the Na(+) gradient to power the transport mechanism.

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

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Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

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