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, Glt(Ph), 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 Glt(Ph). In order to fully utilize the 
Glt(Ph) structure in functional studies of the human glutamate transporters, it 
is essential to understand the transport mechanism of Glt(Ph) 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 Glt(Ph) 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 Glt(Ph), 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 Glt(Ph) and EAAT1.