Na+ -dependent gate dynamics and electrostatic attraction ensure substrate coupling in glutamate transporters

Science Advances
Claudia AllevaJan-Philipp Machtens

Abstract

Excitatory amino acid transporters (EAATs) harness [Na+], [K+], and [H+] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na+ ions, [Na+] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na+:substrate coupling in the EAAT homolog GltPh A lipidic cubic phase x-ray crystal structure of wild-type, Na+-only bound GltPh at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na+ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na+-stabilized gate opening and electrostatic coupling of aspartate to Na+ binding provides a constant Na+:substrate transport stoichiometry over a broad range of neurotransmitter concentrations.

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Citations

Feb 16, 2021·Neurochemical Research·Claudia AllevaChristoph Fahlke
Apr 30, 2021·Journal of Chemical Theory and Computation·Andrei Y KostritskiiJan-Philipp Machtens
May 5, 2021·Chembiochem : a European Journal of Chemical Biology·Kim BartelsChristian Löw
Jun 19, 2021·Communications Biology·Gianluca TrincoDirk J Slotboom
Aug 5, 2021·Neurochemical Research·Solveig G SchmidtClaus J Loland
Nov 9, 2021·The EMBO Journal·Juan C Canul-TecNicolas Reyes

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Methods Mentioned

BETA
x-ray crystallography
X-ray
electron microscopy
fluorescence spectroscopy
fluorescence resonance
PCA
size exclusion chromatography

Software Mentioned

MODELLER
Aimless
membed
trj
cavity
XDS
MOLREP
Coot
GROmaρs
GROMACS

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