Glutamate transport is coupled to the co-transport of 3Na(+) and 1H(+) and the 
countertransport of 1 K(+). However, the mechanism of how this process occurs is 
not well understood. The crystal structure of an archaeal homolog of the human 
glutamate transporters, Glt(Ph), has provided the framework to begin to 
understand the mechanism of transport. The glutamate transporter EAAT2 is 
different from other subtypes in two respects. First, Li(+) cannot support 
transport by EAAT2, whereas it can support transport by the other excitatory 
amino acid transporters, and second, EAAT2 is sensitive to a wider range of 
blockers than other subtypes. We have investigated the relationship between the 
cation driving transport and whether the glutamate analogues, 
l-anti-endo-3,4-methanopyrrolidine-dicarboxylic acid (MPDC) and 
(2S,4R)-4-methylglutamate (4MG), are substrates or blockers of transport. We 
have also investigated the molecular basis for these differences. EAAT2 has a 
Ser residue at position 441 with hairpin loop 2, whereas the corresponding 
residue in EAAT1 is a Gly residue. We demonstrate that if the transporter has a 
Ser residue at this position, then 4MG and MPDC are poor substrates in Na(+), 
and Li(+) cannot support transport of any substrate. Conversely, if the 
transporter has a Gly residue at this position, then in Na(+) 4MG and MPDC are 
substrates with efficacy comparable with glutamate, but in Li(+) 4MG and MPDC 
are poor substrates relative to glutamate. This Ser/Gly residue is located 
between the bound substrate and one of the cation binding sites, which provides 
an explanation for the coupling of substrate and cation binding.