Glutamate transport - relative rates of net uptake and heteroexchange by Zhou Yun 周 云 Master thesis Programme for Cell Biology Department of Molecular Biosciences University of Oslo 1 Table of contents TABLE OF CONTENTS ABSTRACT ACKNOWLEDGEMENTS ABBREVIATIONS INTRODUCTION METABOLISM OF GLUTAMATE GLUTAMATE TRANSPORTERS GLUTAMATE TRANSPORTER STRUCTURE MECHANISM OF GLUTAMATE UPTAKE ANION CONDUCTANCE IN GLUTAMATE TRANSPORTERS TWO DIFFERENT MODES OF SUBSTRATE TRANSLOCATION (EXCHANGE AND NET UPTAKE) WHICH EAAT-SUBTYPE AND WHICH CELLULAR COMPONENT IS RESPONSIBLE FOR MOST BRAIN GLUTAMATE UPTAKE MATERIALS AND METHODS MATERIALS ANIMALS GEL FILTRATION PREPARATION OF RECONSITUTION MIXTURE RECONSTITUTION OF GLUTAMATE TRANSPORTERS INTO LIPOSOMES UPTAKE REACTION FOR RADIOACTIVE AMINO ACID FLUORESCENCE MEASUREMENT RESULTS TEST OF RECONSITUTED TRANSPORTERS UNDER CONDITIONS FAVOURING NET UPTAKE OR HETEROCHANGE LEAKAGE OF GLUTAMATE FROM THE LIPOSOMES UNDERESTIMATION OF THE RELATIVE RATE OF NET UPTAKE IMPORTANCE OF ANIONS D-ASPARTATE VERSUS L-GLUTAMATE RATES OF EXCHANGE AND NET UPTAKE AT SHORTER INCUBATION TIMES AND HIGHER EXTERNAL SUBSTRATE CONCENTRATIONS EFFECTS OF PCBS AND ARACHIDONIC ACID ON TRANSPORTER FUNCTION 2 DISCUSSION THE ADVANTAGE OF THE LIPOSOME ASSAY THE INTEGRITY OF THE LIPOSOME WITH RESPECT TO GLUTAMATE TRANSPORT-ASSOCIATED CHARGE TRANSFER AFFECTS NET UPTAKE AND EXCHANGE DIFFERENTLY THE IMPORTANCE OF THE COMPOSITION OF THE LIPID MEMBRANE FOR TRANSFORTER FUNCTION WHY TERMINALS IN HIPPOCAMPAL SLICES TAKE UP AS MUCH EXTERNAL SUBSTRATE AS GLIA DURING IN VITRO INCUBATION WITH SUBSTRATE IN SPITE OF FEWER TRANSPORTERS CONCLUSION REFERENCES 3 Abstract Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS), and is inactivated by cellular uptake, mostly catalyzed by glutamate (excitatory amino acid) transporter subtype number 2 (EAAT2). EAAT2 protein is mostly found in astroglia (>80%), but there is also some in axon terminals (about 10 %). However, glia and nerve terminals in hippocampal slice preparations accumulate D-aspartate (D-Asp; an EAAT2 substrate) with similar rates when incubated in vitro. This implies that there is an unexplained mismatch between the distribution of EAAT2 transporter protein and the distribution of transport activity. The main aim of the present study has been to find out if the disproportionately high rate of uptake into terminals can be explained by differences in the relative rates of net uptake and of heteroexchange. To do this, glutamate transporters were solubilized and reconstituted in artificial cell membranes (liposomes), and the liposomes were tested for their usefulness as a model: Uptake of external substrate required either internal K+ or internal Na+ and glutamate, and liposomes that were preloaded with glutamate were sufficiently tight to keep most of the internal glutamate for the duration of the assay. In agreement with the notion that the uptake is relatively robust to changes in the lipid environment, addition of polychlorinated biphenyls (PCBs) had no effect, while arachidonic acid inhibited exchange similar to net uptake. Uptake by K+-loaded liposomes was stimulated by addition of a K+ ionophore (valinomycin), but the combination of permeant anions and valinomycin appeared to cause rapid dissipation of driving forces. When the liposomes were studied in the presence of valinomycin, K+-loaded liposomes performed better than liposomes preloaded with Na+ and glutamate, suggesting that net uptake is faster; at least at non-saturating substrate concentrations (< 5 μM). In conclusion, the findings may imply that D-Asp uptake into terminals in hippocampal slice preparations is due to net uptake, and that direct uptake into terminals is more important than currently recognized. 4 Acknowledgements I would like to thank my supervisors Professor Niels Christian Danbolt at The Institute of Basic Medical Sciences, UiO, and Professor Olav Sand at The Department of Molecular Biosciences, UiO. I am very grateful to my internal supervisor Professor Olav Sand and student adviser Torill Rørtveit for providing me the opportunity to learn more about neuroscience. As a member in Niels C. Danbolt’s group, I have obtained basic knowledge about neurotransporters and learnt laboratory practices under the enthusiastic teaching by Professor Niels C. Danbolt. More importantly, I have learnt the attitudes as a researcher in science here. It has been both educational and fun to be a member of a research group with so many gifted colleagues. I would also like to thank Nina Julia Grutle for our collaboration, and Silvia Holmseth and Knut P. Lehre for the assistance. I am very grateful to my parents for supporting me being here. Also, I would like to thank my friend Joseph Fredrick for the encouragement. The work included in this thesis has been performed at the Department of Anatomy of the Institute of Basic Medical Sciences, University of Oslo. 5 Abbreviations AA: arachidonic acid CNS: central nervous system DHK: dihydrokainic acid DMSO: dimethylsulfoxide DTT: dithiothreitol EAAC1: excitatory amino acid carrier 1 (glutamate transporter) (Kanai and Hediger, 1992) EAAT: excitatory amino acid transporter (glutamate transporter) GABA: gamma-aminobutyric acid GDH: L-glutamic dehydrogenase GLAST: glutamate-aspartate transporter (glutamate transporter) (Storck et al., 1992) GLT1: glutamate transporter 1 (Pines et al., 1992) HEPES: N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid KPi: potassium phosphate buffer with pH 7.4 NaPi: sodium phosphate buffer with pH 7.4 PAG: phosphate activated glutaminase PCB: polychlorinated biphenyl PMSF: phenylmethanesulfonyl fluoride PMB-TBOA: PMB-threo-beta-benzyloxyasparate (Shimamoto et al., 1998) 6 Introduction In the mammalian central nervous system (CNS), glutamate is the major excitatory neurotransmitter acting on a number of different receptors (both ionotropic and metabotropic). The receptors are widely expressed, and one or more receptor subtype is found on most (if not all) cell surfaces in the CNS. As the receptors can only be activated by glutamate from the extracellular side, it follows that glutamate is inactive from a transmitter point of view as long as it is intracellular. Consequently, maintenance of a low extracellular concentration is essential for normal synaptic transmission and for protecting cells from the excessive activation of glutamate receptors, known as "excitotoxicity" (for review, see Danbolt, 2001; Ryan and Vandenberg, 2005). This, however, is not a trivial matter considering the large amounts of glutamate in brain tissue, in the order of 10 mmol per kg tissue (for review, see Danbolt, 2001). The highest concentrations are found inside nerve terminals (Ottersen et al., 1992), whereas the concentrations in the extracellular fluid are normally around 3-4 μM (for review, see Danbolt, 2001). This means that the concentration gradient of glutamate across the plasma membranes is several thousand fold. Extracellular glutamate can only be removed by cellular uptake catalyzed by the glutamate transporter proteins (for review, see Danbolt, 2001). Metabolism of glutamate The metabolism of glutamine and glutamate in the brain is compartmentalized (Fig. 1). Glutamate is taken up into astrocytes after synaptic release, and is converted to glutamine by means of the astrocyte-specific enzyme glutamine synthetase (for review, see Broman et al., 2000). Glutamine does not have the ability to activate glutamate receptors, and can be safely released to the extracellular fluid via the system N glutamine transporter SN1 (Chaudhry et al., 1999). Extracellular glutamine is taken up by neurons by specific glutamine transporters (e.g. SAT1 and SAT2) and converted to glutamate by means of phosphate-activated glutaminase (PAG) or used in other metabolic processes (for review, see McKenna et al., 2000; Danbolt, 2001). 7 Fig. 1. The glutamate-glutamine cycle. Glutamate is released from nerve-endings and also from astroglia (not shown). After release, it is taken up by astroglia (1), by terminals (2) and by dendritic spines (3). The relative importance of 1 - 3 is discussed in the main text. Astroglia converts glutamate to glutamine and releases it (5). Terminals take up glutamine (6), convert it to glutamate and pack it into synaptic vesicles (4). From Danbolt, 2001. Glutamate transporters There are several transporter proteins that are able to translocate glutamate through membranes (for review, see Danbolt, 2001). These include intracellular transporters in mitochondria ("mitochondrial glutamate transporters": aralar1; citrin; GC1 and GC2; genes slc25a12, slc25a13, slc25a22 and slc25a18, respectively) and in synaptic vesicles ("vesicular glutamate transporters": VGLUT1-3; genes slc17a6, slc17a7 and slc17a8). In the plasma membrane, other transporters are found. These include transporters for neutral amino acids (e.g. ASCT2; gene slc1a5) and for dicarboxylates (e.g. NaC3; gene slc13a5), which are able to transport glutamate with low affinity. There is also a glutamate-cystine exchanger (xCT; gene slc7a11). In spite of this, the term "glutamate transporter" (or "high affinity glutamate transporter") is usually used as a synonym for the five "Excitatory Amino Acid Transporters" (EAATs): EAAT1 (GLAST; Storck et al., 1992; gene: slc1a3), EAAT2 (GLT1; Pines et al., 1992; gene: slc1a2), EAAT3 (EAAC1; Kanai and Hediger, 1992; gene: slc1a1), EAAT4 (Fairman et al., 1995; gene: slc1a6), and EAAT5 (Arriza et al., 1997; gene: slc1a7). These transporter