%0 Journal Article
%~ PubMed
%A Carland, J E
%A Mansfield, R E
%A Ryan, R M
%A Vandenberg, R J
%T Oleoyl-L-Carnitine Inhibits Glycine Transport by GlyT2.
%B British Journal of Pharmacology
%D 2013
%C United Kingdom
%I John Wiley & Sons Ltd.
%V 168
%N 4
%P 891-902
%@ 0007-1188
%X 
%Z FOR Codes: 111501 110902 60110


%0 Journal Article
%~ PubMed
%A Bastug, Turgut
%A Heinzelmann, Germano
%A Kuyucak, Serdar
%A Salim, Marietta
%A Vandenberg, Robert J
%A Ryan, Renae M
%T Position of the third Na+ site in the aspartate transporter GltPh and the human glutamate transporter, EAAT1.
%B PloS One
%D 2012
%C United States
%I Public Library of Science
%V 7
%N 3
%P e33058
%@ 1932-6203
%X 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.
%Z FOR Codes: 806


%0 Journal Article
%~ PubMed
%A Bailey, Charles G
%A Ryan, Renae M
%A Thoeng, Annora D
%A Ng, Cynthia
%A King, Kara
%A Vanslambrouck, Jessica M
%A Auray-Blais, Christiane
%A Vandenberg, Robert J
%A Bröer, Stefan
%A Rasko, John E J
%T Loss-of-function mutations in the glutamate transporter SLC1A1 cause human dicarboxylic aminoaciduria.
%B The Journal of clinical investigation
%D 2011
%C United States
%I American Society for Clinical Investigation
%V 121
%N 1
%P 446-53
%@ 1558-8238
%X Solute carrier family 1, member 1 (SLC1A1; also known as EAAT3 and EAAC1) is the major epithelial transporter of glutamate and aspartate in the kidneys and intestines of rodents. Within the brain, SLC1A1 serves as the predominant neuronal glutamate transporter and buffers the synaptic release of the excitatory neurotransmitter glutamate within the interneuronal synaptic cleft. Recent studies have also revealed that polymorphisms in SLC1A1 are associated with obsessive-compulsive disorder (OCD) in early-onset patient cohorts. Here we report that SLC1A1 mutations leading to substitution of arginine to tryptophan at position 445 (R445W) and deletion of isoleucine at position 395 (I395del) cause human dicarboxylic aminoaciduria, an autosomal recessive disorder of urinary glutamate and aspartate transport that can be associated with mental retardation. These mutations of conserved residues impeded or abrogated glutamate and cysteine transport by SLC1A1 and led to near-absent surface expression in a canine kidney cell line. These findings provide evidence that SLC1A1 is the major renal transporter of glutamate and aspartate in humans and implicate SLC1A1 in the pathogenesis of some neurological disorders.
%Z FOR Codes: 60110 60412 110101


%0 Journal Article
%~ PubMed
%A Vandenberg, Robert J
%A Handford, Cheryl A
%A Campbell, Ewan M
%A Ryan, Renae M
%A Yool, Andrea J
%T Water and urea permeation pathways of the human excitatory amino acid transporter EAAT1.
%B The Biochemical journal
%D 2011
%C United Kingdom
%I Portland Press Ltd.
%V 439
%N 2
%P 333-40
%@ 1470-8728
%X Glutamate transport is coupled to the co-transport of 3 Na+ and 1 H+ followed by the counter-transport of 1 K+. In addition, glutamate and Na+ binding to glutamate transporters generates an uncoupled anion conductance. The human glial glutamate transporter EAAT1 (excitatory amino acid transporter 1) also allows significant passive and active water transport, which suggests that water permeation through glutamate transporters may play an important role in glial cell homoeostasis. Urea also permeates EAAT1 and has been used to characterize the permeation properties of the transporter. We have previously identified a series of mutations that differentially affect either the glutamate transport process or the substrate-activated channel function of EAAT1. The water and urea permeation properties of wild-type EAAT1 and two mutant transporters were measured to identify which permeation pathway facilitates the movement of these molecules. We demonstrate that there is a significant rate of L-glutamate-stimulated passive and active water transport. Both the passive and active L-glutamate-stimulated water transport is most closely associated with the glutamate transport process. In contrast, L-glutamate-stimulated [14C]urea permeation is associated with the anion channel of the transporter. However, there is also likely to be a transporter-specific, but glutamate independent, flux of water via the anion channel.
%Z FOR Codes: 111501 110902 60105


%0 Book Section
%A McKinzie, Audra
%A Ryan, Renae
%A Vandenberg, Robert
%T Site-directed mutagenesis in the study of membrane transporters.
%B Methods in Molecular Biology: Membrane Transporters in Drug Discovery and Development
Methods and Protocols
%D 2010
%C United States
%I Humana Press
%V 
%N 
%P 277-293
%@ 9781607616993
%E Yan, Qing
%X 
%Z FOR Codes: 601 1110 1111


%0 Journal Article
%~ PubMed
%A Ryan, Renae M
%A Kortt, Nicholas C
%A Sirivanta, Tan
%A Vandenberg, Robert J
%T The position of an arginine residue influences substrate affinity and K coupling in the human glutamate transporter, EAAT1.
%B Journal of Neurochemistry
%D 2010
%C United Kingdom, United States
%I Wiley-Blackwell Publishing Ltd.
%V 114
%N 2
%P 565-575
%@ 0022-3042
%X Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and extracellular glutamate levels are controlled by a family of transporters known as excitatory amino acid transporters (EAATs). The EAATs transport glutamate and aspartate with similar micromolar affinities and this transport is coupled to the movement of Na(+), K(+), and H(+). The crystal structure of a prokaryotic homologue of the EAATs, aspartate transporter from Pyrococcus horokoshii (Glt(Ph)), has yielded important insights into the architecture of this transporter family. Glt(Ph) is a Na(+)-dependent transporter that has significantly higher affinity for aspartate over glutamate and is not coupled to H(+) or K(+). The highly conserved carboxy-terminal domains of the EAATs and Glt(Ph) contain the substrate and ion binding sites, however, there are a couple of striking differences in this region that we have investigated to better understand the transport mechanism. An arginine residue is in close proximity to the substrate binding site of both Glt(Ph) and the EAATs, but is located in transmembrane domain (TM) 8 in the EAATs and hairpin loop 1 (HP1) of Glt(Ph). Here we report that the position of this arginine residue can explain some of the functional differences observed between the EAATs and Glt(Ph). Moving the arginine residue from TM8 to HP1 in EAAT1 results in a transporter that has significantly increased affinity for both glutamate and aspartate and is K(+) independent. Conversely, moving the arginine residue from HP1 to TM8 in Glt(Ph) results in a transporter that has reduced affinity for aspartate.
%Z FOR Codes: 60108 1109 1701


%0 Journal Article
%~ PubMed
%A Edington, Amelia R
%A McKinzie, Audra A
%A Reynolds, Aaron J
%A Kassiou, Michael
%A Ryan, Renae M
%A Vandenberg, Robert J
%T Extracellular loops 2 and 4 of GLYT2 are required for N-arachidonyl-glycine inhibition of glycine transport.
%B The Journal of biological chemistry
%D 2009
%C United States
%I American Society for Biochemistry and Molecular Bi
%V 284
%N 52
%P 36424-30
%@ 1083-351X
%X Concentrations of extracellular glycine in the central nervous system are regulated by Na(+)/Cl(-)-dependent glycine transporters, GLYT1 and GLYT2. N-Arachidonylglycine (NAGly) is an endogenous inhibitor of GLYT2 with little or no effect on GLYT1 and is analgesic in rat models of neuropathic and inflammatory pain. Understanding the molecular basis of NAGly interactions with GLYT2 may allow for the development of novel therapeutics. In this study, chimeric transporters were used to determine the structural basis for differences in NAGly sensitivity between GLYT1 and GLYT2 and also the actions of a series of related N-arachidonyl amino acids. Extracellular loops 2 and 4 of GLYT2 are important in the selective inhibition of GLYT2 by NAGly and by the related compounds N-arachidonyl-gamma-aminobutyric acid and N-arachidonyl-d-alanine, whereas only the extracellular loop 4 of GLYT2 is required for N-arachidonyl-l-alanine inhibition of transport. These observations suggest that the structure of the head group of these compounds is important in determining how they interact with extracellular loops 2 and 4 of GLYT2. Site-directed mutagenesis of GLYT2 EL4 residues was used to identify the key residues Arg(531), Lys(532), and Ile(545) that contribute to the differences in NAGly sensitivity.
%Z FOR Codes: 110902 111501 60110


%0 Journal Article
%~ PubMed
%A Huang, Shiwei
%A Ryan, Renae M
%A Vandenberg, Robert J
%T The role of cation binding in determining substrate selectivity of glutamate transporters.
%B The Journal of biological chemistry
%D 2009
%C United States
%I Amer Soc Biochemistry Molecular Biology Inc
%V 284
%N 7
%P 4510-5
%@ 1083-351X
%X 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.
%Z FOR Codes: 111501 110902


%0 Journal Article
%~ PubMed
%A Vandenberg, Robert J
%A Huang, Shiwei
%A Ryan, Renae M
%T Slips, leaks and channels in glutamate transporters.
%B Channels
%D 2008
%C United States
%I Landes Bioscience
%V 2
%N 1
%P 51-58
%@ 1933-6969
%X Glutamate transporters are unusual proteins in that they can function as both a transporter and a chloride channel. With the recent determination of the crystal structure of an archaeal aspartate transporter it is now possible to begin to put together a physical picture of how these proteins are able to carry out their dual functions. In this review we shall discuss our current understanding of the functional states of glutamate transporters and how they may arise. We will also discuss some of the alternate conducting states of glutamate transporters and provide definitions of the various states.
%Z FOR Codes: 110902 111501 111601


%0 Journal Article
%~ PubMed
%A Boudker, Olga
%A Ryan, Renae M
%A Yernool, Dinesh
%A Shimamoto, Keiko
%A Gouaux, Eric
%T Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter.
%B Nature
%D 2007
%C 175 Fifth Ave, New Y
%I Springer-Verlag
%V 445
%N 7126
%P 387-393
%@ 1476-4687
%X Secondary transporters are integral membrane proteins that catalyse the movement of substrate molecules across the lipid bilayer by coupling substrate transport to one or more ion gradients, thereby providing a mechanism for the concentrative uptake of substrates. Here we describe crystallographic and thermodynamic studies of Glt(Ph), a sodium (Na+)-coupled aspartate transporter, defining sites for aspartate, two sodium ions and d,l-threo-beta-benzyloxyaspartate, an inhibitor. We further show that helical hairpin 2 is the extracellular gate that controls access of substrate and ions to the internal binding sites. At least two sodium ions bind in close proximity to the substrate and these sodium-binding sites, together with the sodium-binding sites in another sodium-coupled transporter, LeuT, define an unwound alpha-helix as the central element of the ion-binding motif, a motif well suited to the binding of sodium and to participation in conformational changes that accompany ion binding and unbinding during the transport cycle.
%Z FOR Codes: 110999