Identification of Functional Domains of the Human Glutamate Transporters EAAT1 and EAAT2

Abstract

Glutamate transporters serve the important function of mediating removal of glutamate released at excitatory synapses and maintaining extracellular concentrations below excitotoxic levels. Excitatory amino acid transporter subtypes EAAT1 and EAAT2 have a high degree of sequence homology and similar predicted topology and yet display a number of functional differences. Several recombinant chimeric transporters were generated to identify domains that contribute to functional differences between EAAT1 and EAAT2. Wild-type transporters and chimeric transporters were expressed in Xenopus laevis oocytes, and electrogenic transport was studied under voltage clamp conditions. The differential sensitivity of EAAT1 and EAAT2 to transport blockers, kainate, threo-3-methylglutamate, and (2S,4R)-4-methylglutamate as well as L-serine-O-sulfate transport and chloride permeability were employed to characterize chimeric transporters. One particular region, transmembrane domains 9 and 10, plays an important role in defining these functional differences. The intracellular carboxyl-terminal region may also play a minor role in conferring an effect on chloride permeability. This study provides important insight into the identification of functional domains that determine differences among glutamate transporter subtypes.L-Glutamate transport in the central nervous system and also in peripheral organs is mediated by a family of structurally related membrane proteins. In the central nervous system, glutamate transporters play an important role by mediating removal of glutamate released at excitatory synapses and also influencing the kinetics of glutamate receptor activation (1, 2). Complementary DNAs encoding five different glutamate transporters have been identified (3-7). The human clones have been named Excitatory Amino Acid Transporters 1-5 (EAAT1-5) (6-8). The recent cloning and expression of recombinant glutamate transporter proteins in heterologous expression systems allow a number of questions concerning the molecular basis for transporter function to be addressed. In particular, an important aspect of such studies is the identification of domains involved in substrate recognition and substrate translocation through the membrane. In this study we have constructed a series of chimeric glutamate transporters, using the EAAT1 and EAAT2 subtypes, and exploited the pharmacological and electrophysiological differences between the two subtypes to map functional domains of these proteins.To make meaningful predictions about the functional role of various domains, an understanding of the structure of the transporters is particularly useful. The predicted amino acid sequences of EAAT1 and EAAT2 are 65% identical, and if conservative substitutions are allowed the degree of relatedness is increased to 80% (8). These two proteins are likely to form very similar structures and therefore are good candidates for the generation of functional chimeras. Through the use of hydrophobicity analysis of the amino acid sequences it has been predicted t...

Two serine residues of the glutamate transporter GLT-1 are crucial for coupling the fluxes of sodium and the neurotransmitter

“…Although this scenario cannot be ruled out we favor the simple explanation that position 440 is not far from the substratebinding site. A recent study employing chimeric glutamate transporters identifies one domain as particularly important for substrate binding (40). It is of special interest to note that serine residues 440 and 443 are at the amino-terminal edge of this domain.…”

A conserved serine-rich stretch in the glutamate transporter family forms a substrate-sensitive reentrant loop

“…This serine-rich motif is located in the putative cytoplasmic loop between membrane-spanning helices 6 and 7 in the C-terminal half of the transporters, which is thought to contain the translocation path and the binding sites for glutamate and the coupling ions (6)(7)(8)(9)(10)(11)(12)(13). The loop comprising the serine-rich motif was studied by cysteine-scanning mutagenesis in the glutamate transporter GltT of the bacterium B. stearothermophilus.…”

“…The glutamate transporters belong to a large family of transport proteins in which bacterial glutamate transporters also are found, including GltT of Bacillus stearothermophilus (5). Evidence is accumulating that the C-terminal half of the transporters, which is particularly well conserved, constitutes a major part of the translocation pathway and contains the binding sites for the substrate and cotransported ions (6)(7)(8)(9)(10)(11)(12)(13). This part of the transporters contains a serine-rich sequence motif, which is conserved in all members of the enormously divergent family.…”

Cysteine-scanning Mutagenesis Reveals a Highly Amphipathic, Pore-lining Membrane-spanning Helix in the Glutamate Transporter GltT

“…Three hydrophilic stretches are considerably longer in the eukaryotic proteins: the amino-terminal and carboxyl-terminal extensions and the region between the third and fourth membrane-spanning segment, which is glycosylated in the eukaryotic members. Evidence is accumulating that the carboxyl-terminal half of the transporters, which is particularly well conserved, constitutes a major part of the translocation pathway and contains the binding sites for the substrate and cotransported ions (7,(12)(13)(14)(15)(16)(17)(18). The bacterial and eukaryotic glutamate transporters have different coupling ion specificity.…”