Abstract
Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft. Here, we develop and apply high-speed atomic force microscopy line-scanning (HS-AFM-LS) combined with automated state assignment and transition analysis for the determination of transport dynamics of unlabeled membrane-reconstituted GltPh, a prokaryotic EAAT homologue, with millisecond temporal resolution. We find that GltPh transporters can operate much faster than previously reported, with state dwell-times in the 50 ms range, and report the kinetics of an intermediate transport state with height between the outward- and inward-facing states. Transport domains stochastically probe transmembrane motion, and reversible unsuccessful excursions to the intermediate state occur. The presented approach and analysis methodology are generally applicable to study transporter kinetics at system-relevant temporal resolution.
Highlights
Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft
Excitatory amino acid transporters (EAATs) are a family of integral membrane proteins that are essential in the mammalian central nervous system (CNS)
The dense packing might be a consequence of the chosen lipid composition that is different from the native environment in Pyrococcus horikoshii[38], we note that mammalian EAATs cluster too with up to ~5000 transporters/μm[2] in glial membranes around synaptic clefts[39,40]
Summary
Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft. 1234567890():,; Excitatory amino acid transporters (EAATs) are a family of integral membrane proteins that are essential in the mammalian central nervous system (CNS) This family of proteins assures efficient neurotransmission and prevents glutamate-mediated neurotoxic effects by regulating extracellular transmitter levels of neurons and glial cells surrounding synapses. Expression of glutamate transporters is not exclusive to the CNS, and occurs in other organs such as kidney (EAAT3) or the retina where EAAT5 mediates glutamate-dependent chloride influx into cells[5,6,7] This vital family of transporters symports three sodium (Na+) ions and one proton (H+) along with the EAA, and antiports one potassium (K+) ion[8,9]. While other transporters function by the rocker-switch (e.g., LacY25) or gated-pore (e.g., LeuT26) mechanisms, GltPh displays an “elevator” mechanism, whereby the transport domains move ∼1.8 nm across the membrane[12,27,28,29,30,31,32]
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