Glutamate transporter : a brief history of excitatory amino acid transmembrane proteins

The history of glutamate transporters starts with its ligand


In 1866, in Germany, working on wheat plant proteins, Ritthausen succeeded in crystallizing a new molecule, glutamic acid HOOC(CH2)2CH(NH2)-COOH (α-aminoglutaric acid, C5H9NO4), or glutamate for its anionic form. Given the technical means available, many of us still consider Ritthausen's work as simply prodigious for its time (1).


And curiously enough, nobody was interested in this molecule for years.


Or should I say decades.


It was in 1920 that Thunberg reused the substance to work on oxidative processes. He realized that glutamic acid differs from most of the amino acids, because it is oxidized in organs whose metabolism is supposed to concern mainly carbohydrates and which are quite inert towards most of the other amino acids (2). The discovery of this thin permeable membrane between amino acid and carbohydrate metabolism gave rise by extension to the Krebs cycle in 1937, after the intervention of the important inputs from Needham.


As early as 1936, Hans Weil-Malherbe took care to review all the work of his predecessors and began to establish a hypothetical link between glutamic acid and the nervous system. Many years later, Hans became one of the pioneers of glutamic acid therapy in mental deficiencies and epileptic disorders, and perhaps one of the first to make neurochemistry a science in its own right.


But it was on the other side of the globe, that Weil-Malherbe 's work resonated the most. In Tokyo, at Keio University, Hayashi applied various doses of sodium glutamate into grey matters of motor cortex and circulation in dogs to observe convulsions and salivary reflexes, demonstration of a specific and direct excitatory action of glutamate on the mammalian on the central nervous system. Nevertheless, Hayashi's work does not really allow reaching a conclusion on the links between glutamate and GABA and on the fact that they are natural neurotransmitters (3).


It will be necessary to wait for the work of Krnjevic and Phillis in 1963 to be convinced that glutamate and GABA are natural transmitters in the brain as they pointed out that all neurones could be excited with L-glutamate and certain related dicarboxylic amino acids (4).


And we know now for sure that glutamate is the major neurotransmitter involved in neuronal excitation, and so part of excitatory amino acids (EAA) family. Nevertheless, fully understanding how this neurotransmitter is transported from one place to another in the cell is another matter.


Glutamate protein dual ion channel/transport behavior: a complex transmembrane receptor


In their early descriptions of the excitatory activity of a wide variety of acidic amino acids, Curtis & Watkins were the first to put forward the idea of a "three-point attachment" receptor model, with which the charged groups of the anionic glutamate molecule interact. This interaction causes a conformational change in the receptor and associated membrane molecules, opening the pores to allow extracellular sodium ions to flow along their electrochemical gradient and depolarize the cell (5).


While ion channels are non-saturable and oscillate between two distinct and reversible states (open and closed) defined by thermodynamic probabilities, transporters must change their conformation to move the molecule across the cell membrane. Unlike ion channels, transporters are saturable and not governed by equilibrium laws.


And in the case of the glutamate receptor, we are indeed in the presence of both a transporter and an ion channel (a rather rare characteristic that is nevertheless found in other receptors of the "SLC" family of solute transporters).


Localized to the plasma membrane of neurons and astrocytes, transmembrane glutamate receptors are called excitatory amino acid transporters or EAATs. There are five different subtypes, named EAAT1 to EAAT5, and all present the unusual structural pattern of eight membrane-spanning domains and two reentrant loops of opposite orientation.


In rodents, the human excitatory amino acid transporter 2 (EAAT2) is designated as GLT-1. GLT-1 is the major glutamate transporter in the central nervous system (CNS) where it is predominantly expressed by astrocytes (Danbolt, 2001).


SYnAbs generation of rat-LOU monoclonals anti-splice variant GLT-1b glutamate receptor


GLT-1 has two alternatively spliced variants: GLT-1a and GLT-1b, whose intracellular C-terminus differs by a single amino acid. Since the functions and regulations of these isoforms in the face of environmental constraints remained unclear, SYnAbs addressed this issue (6).


Yannick Nizet immunized LOU/C rats, a proprietary species of SYnAbs well known to be an excellent responder to haptens, and which has the particularity of being able to produce antibodies against self-antigens. The choice of the immunogen was the synthetic peptide H2N-GPFPFLDIETCI-COOH corresponding to the C-terminal sequence of GLT-1b.


Hybridomas were produced by fusing the SYnAbs proprietary non-secreting LOU rat cell line IR983F with splenocytes from one of the immunized LOU/C rats whose serum specifically recognized the peptide. Limit dilutions of the hybridoma cells allowed isolation of clones, and the specificity of the antibody secreted by the individual clones was confirmed by ELISA against the synthetic peptide and excluded against the non-relevant peptide encoding GLT-1a.


The secondary antibody was conjugated to HRP, and this mouse monoclonal anti-rat IgG was none other than MARG diluted 1:4000, off-the-shelf reference of SYnAbs catalog.


(1) Ritthausen, H. Ueber die Glutaminsäure. J. für Prakt. Chemie 99, 454–462 (1866).

(2) Hans Weil-Malherbe. Studies on brain metabolism: The metabolism of glutamic acid in brain. The biochemical journal. Volume 30, Issue 4, April 1936.

(3) Takashi Hayashi. Effects of sodium glutamate on the nervous system. Keio J. Med. 1954;3:192–193.

(4) Krnjevic, K. and Phillis, J.W. (1963) Iontophoretic Studies of Neurons in the Mammalian Cerebral Cortex. The Journal of Physiology, 165, 274-304.

(5) Curtis D.R., Watkins J.C. The excitation and depression of spinal neurones by structurally related amino acids. J. Neurochem.

(6) Focant, Marylène C. ; Goursaud, Stéphanie ; Nizet, Yannick ; Hermans, Emmanuel. Differential regulation of C-terminal splice variants of the glutamate transporter GLT-1 by tumor necrosis factor-alpha in primary cultures of astrocytes.  In: Neurochemistry International : the journal for the publication of cellular and molecular aspects of neurochemistry, Vol. 58, no. 7, p. 751-758 (2011)