Abstract
Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na+ ions, one H+ and the counter-transport of one K+ ion. Transport by an archaeal homologue of the human glutamate transporters, GltPh, whose three dimensional structure is known is also coupled to three Na+ ions but only two Na+ ion binding sites have been observed in the crystal structure of GltPh. In order to fully utilize the GltPh structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of GltPh and accurately determine the number and location of Na+ ions coupled to transport. Several sites have been proposed for the binding of a third Na+ ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for GltPh and reveal a new site for the third Na+ ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in GltPh, and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na+ compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na+ ion in GltPh and EAAT1.
Highlights
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system
Glutamate transport via the excitatory amino acid transporters (EAATs) is coupled to the cotransport of three Na+ ions and one H+ ion followed by the counter-transport of one K+ ion [3,4]
Molecular dynamics is a powerful tool that can simulate movements and binding events from static snapshots of proteins obtained from X-ray crystallography, but functional studies are vital to confirm these predictions and maximise the information obtained from molecular dynamics (MD) simulations
Summary
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. The extracellular concentration of glutamate is predicted to be as low as 25 nM [1] and is maintained by specific transport proteins called excitatory amino acid transporters (EAATs). Excessive extracellular glutamate is toxic for neurons, and its concentration needs to be strictly controlled. Loss of this control due to dysfunction of EAATs has been implicated in several neurological diseases such as Alzheimer’s disease, motor neuron disease and amyotrophic lateral sclerosis [2]. Glutamate transport via the EAATs is coupled to the cotransport of three Na+ ions and one H+ ion followed by the counter-transport of one K+ ion [3,4]. Determination of the crystal structure of a glutamate transporter homologue from
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