Multimodal dynamics control activity of a glial glutamate transporter.

Membrane transporters move polar solutes across lipid bilayers to regulate cellular metabolism, signaling, and drug distribution. These proteins operate via an alternating-access mechanism, cycling between extracellular-, intermediate-, and intracellular-facing conformations. The human excitatory amino acid transporter 1 (EAAT1) protects neurons from excitotoxic damage by mediating the uptake of glutamate and aspartate into glial cells. Defects in EAAT1 function result in numerous pathologies, including epilepsy and ataxia, suggesting that positive modulation of these transporters might ameliorate glutamate neurotoxicity. However, developing EAAT1 activators requires understanding the timing of conformational changes, which remain largely unexplored. Here, we establish an experimental platform that combines single-molecule Förster resonance energy transfer (smFRET) to monitor real-time conformational dynamics, single-transporter activity assays to correlate dynamics with function, and cryogenic electron microscopy (cryoEM) to visualize discrete conformations at high resolution. This platform enables detection of Ångstrom-scale movements of single transporter molecules in real time, revealing that EAAT1 intersperses rapid conformational dynamics with long pauses. Slow and fast dynamics can be modulated by substrates, membrane composition, and mutations, and are correlated with the enrichment of specific structural states. We leverage this platform to investigate an EAAT1 mutation associated with severe episodic ataxia and show that it inhibits transport by stabilizing a paused cytoplasm-facing conformation. These results identify multimodal dynamics as an intrinsic, regulatable feature of EAAT1 function and, therefore, a potential therapeutic target. Henceforth, our integrated platform will facilitate investigations of other regulatory factors, including the effects of small-molecule and lipid modulators on the transport cycle.