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.
Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous GltPh coordinates L-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na(+) over Li(+). S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for L-aspartate over D-aspartate and L-glutamate, and lost their selectivity for Na(+) over Li(+). Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.
From the Institute of Biochemistry and Molecular Medicine and Swiss National Center of Competence in Research, National Center of Competence in Research (NCCR) TransCure, University of Bern, 3012 Bern, Switzerland asimonin@hotmail.fr.
Alexandre
A
Simonin
From the Institute of Biochemistry and Molecular Medicine and Swiss National Center of Competence in Research, National Center of Competence in Research (NCCR) TransCure, University of Bern, 3012 Bern, Switzerland nim71@pitt.edu.
Nicolas
N
Montalbetti
From the Institute of Biochemistry and Molecular Medicine and Swiss National Center of Competence in Research, National Center of Competence in Research (NCCR) TransCure, University of Bern, 3012 Bern, Switzerland.
Gergely
G
Gyimesi
From the Institute of Biochemistry and Molecular Medicine and Swiss National Center of Competence in Research, National Center of Competence in Research (NCCR) TransCure, University of Bern, 3012 Bern, Switzerland.
Jonai
J
Pujol-Giménez
From the Institute of Biochemistry and Molecular Medicine and Swiss National Center of Competence in Research, National Center of Competence in Research (NCCR) TransCure, University of Bern, 3012 Bern, Switzerland matthias.hediger@ibmm.unibe.ch.
Matthias A
MA
Hediger
D019943:Amino Acid Substitution; D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D027342:Excitatory Amino Acid Transporter 2; D050491:Glutamate Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D008094:Lithium / Q000378:metabolism*; D020125:Mutation, Missense; D009865:Oocytes; D012694:Serine / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D014982:Xenopus laevis
D027342:Excitatory Amino Acid Transporter 2; D050491:Glutamate Plasma Membrane Transport Proteins; C493397:SLC1A2 protein, human; D012694:Serine; D008094:Lithium; D012964:Sodium
GLT-1; SLC1A2; excitatory neurotransmission; excitotoxicity; glutamate transporter; molecular dynamics; mutagenesis; neurotoxicity; neurotransmitter transport; structure-function; structure/function studies