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  • br Excitatory amino acid transporter mediated glutamate tran

    2022-01-14


    Excitatory amino Tranilast transporter-mediated glutamate transport Early studies detailing the presence of uptake systems capable of transporting glutamate and aspartate into neurons and glia represented a critical step in the overall task of demonstrating that l-glutamate was indeed an endogenous excitatory neurotransmitter. Thus, the acceptance of l-glutamate as a neurotransmitter mandated that a means to terminate the excitatory signal be identified. Rapid transport out of the synaptic cleft provided a plausible mechanism, particularly in the absence of a classic degradative enzyme. The presence of this transport activity also served as one of the first biochemical markers of excitatory synapses (Cotman et al., 1981). Two of the properties that emerged from these studies that clearly distinguished the uptake of l-glutamate into CNS preparations (and remain defining qualities today) were a high-affinity for substrate and a requirement for sodium (Logan & Snyder, 1972). Kinetic studies following the transport of radiolabeled l-glutamate or l-aspartate yielded Km values in the 1- to 10-μM range, rather than the mM values more typically associated with epithelial amino acid uptake systems. The observed sodium dependency reflects the driving force for transport, where the movement of sodium and potassium ions down their respective concentrations gradients provides the energy needed to import l-glutamate against its concentration gradient. Current stoichiometric models indicate that EAATs operate via an “alternate access mechanism” whereby the translocation of 1 molecule of l-glutamate into the cell is coupled with the inward co-transport with 3 sodium ions and 1 proton (Zerangue & Kavanaugh, 1996; see Fig. 1). In turn, the return of the empty carrier to the extracellular side of the plasma membrane is coupled to the export of 1 potassium ion. In addition to the movement of these ions, glutamate also appears capable of activating an EAAT-mediated anion current that essentially allows the transporters to function as glutamate-gated chloride channels. This anion current, which is particularly strong in EAAT4 and 5, is sodium- and glutamate-dependent, but not stoichiometrically coupled to the uptake of substrate (Fairman et al., 1995, Arriza et al., 1997, Otis & Kavanaugh, 2000). The activation of this current may contribute to signaling in regions expressing high levels of EAAT4 or EAAT5, such as the retina. The driving force provided by the sodium and potassium ion gradients can maintain intracellular concentrations of l-glutamate that are more than 105-fold greater than those found extracellularly (Zerangue & Kavanaugh, 1996). The proposed stoichiometry is also consistent with the electrogenic nature of EAAT activity, where the uptake of l-glutamate results in the inward movement of positive charge. This also allows the flux of l-glutamate (or other substrates) through the transporter to be followed in real time using electrophysiological recording techniques, an approach that provides a number of advantages over traditional approaches quantifying the flux of radiolabeled substrates (Wadiche et al., 1995a) and multiple chapters in (Amara, 1998). Indeed, more recent studies employing these approaches often report kinetic data for substrates in terms of maximally induced currents (i.e., Imax or % Imax of l-glutamate), rather than classic Vmax values.
    Excitatory amino acid transporter subtypes Early indications that glutamate uptake in the CNS was not mediated by a single homogenous system came to light primarily in comparative pharmacological studies, where the potency of inhibitors were observed to vary among CNS preparations (Roberts & Watkins, 1975, Schousboe & Divac, 1979, Balcar et al., 1987, Robinson et al., 1993b). This issue Tranilast was definitively resolved when, almost simultaneously, 3 different high-affinity, sodium-dependent glutamate transporters were cloned and expressed: GLAST (Storck et al., 1992) and GLT-1 (Pines et al., 1992) from rat brain and EAAC1 (Kanai & Hediger, 1992) from rabbit intestine. This was followed 2 years later by the isolation of the homologous transporters from human brain, which were referred to as excitatory amino acid transporters (EAAT) 1, 2, and 3, respectively (Arriza et al., 1994). (To simplify comparative statements, the EAAT subtype nomenclature will principally be used in this review for all of the transporters, not just the human isoforms.) Since then, screens of cDNA libraries from human cerebellum and retina led to the isolation of EAAT4 (Fairman et al., 1995) and EAAT5 (Arriza et al., 1997), respectively. Sequence comparisons among the 5 EAATs indicate that there is about 50–60% homology among the subtypes and about 30–40% homology with the ASC transporters that are also included in the same gene family (see above; Seal & Amara, 1999).