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

Abstract

The neurotoxicity of glutamate in the central nervous system is restricted by several (Na ؉ ؉ K ؉ )-coupled transporters for this neurotransmitter. The astroglial transporter GLT-1 is the only subtype that exhibits high sensitivity to the nontransportable glutamate analogue dihydrokainate. A marked reduction in sensitivity to the blocker is observed when serine residues 440 and 443 are mutated to glycine and glutamine, which, respectively, occupy these positions in the other homologous glutamate transporters. They are located in the ascending limb of the recently identified pore-loop-like structure. Strikingly, mutation of serine-440 to glycine enables not only sodium but also lithium ions to drive net inf lux of acidic amino acids. Moreover, the efficiency of lithium as a driving ion for glutamate transport depends on the nature of the amino acid residue present at position 443. Mutant transporters containing single cysteines at the position of either serine residue become sensitive to positively as well as negatively charged methanethiosulfonate derivatives. In S440C transporters significant protection against this inhibition is provided both by transportable and nontransportable glutamate analogues, but not by sodium alone. Our observations indicate that the pore-loop-like structure plays a pivotal role in coupling ion and glutamate f luxes and suggest that it is close to the glutamate-binding site.

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

“…5). Similar changes in cation selectivity that were observed for L-glutamate, L-aspartate, and D-aspartate as substrates have already been described for S440G (47). We found that S364D and S364N were not functional ( Figs.…”

Structure of a glutamate transporter homologue from Pyrococcus horikoshii

“…In eukaryotic transporters HP2 contains residues that are important for sodium binding. In particular, Ser 440 and Ser 443 in GLT-1, which are equivalent to Gly 354 and Gly 357 in Glt Ph 7H, are important for sodium selectivity of the transporter 31 . Gly 354 and Gly 357 in Glt Ph 7H flank the tip of HP2 and are within 5 Å of the substrate-binding site.…”

Intersubunit Interactions in EAAT4 Glutamate Transporters

“…We here study glutamate uptake and anion conduction at various concentrations of transporter substrates such as Na ϩ and glutamate. Experiments were performed with wild-type (WT) EAAT4 as well as with mutant transporters bearing two point mutations, G464S and Q467S, that are located in the neighborhood of the glutamate binding pocket (Zhang and Kanner, 1999;Yernool et al, 2004). We demonstrate that several cooperatively interacting glutamate binding sites need to be occupied to activate the anion channel.…”