Glutamate transporter GLAST/EAAT1 directs cell surface expression of FXYD2/γ subunit of Na, K-ATPase in human fetal astrocytes

Abstract

Na⁺-dependent uptake of excitatory neurotransmitter glutamate in astrocytes increases cell energy demands primarily due to the elevated ATP consumption by glutamine synthetase and Na⁺, K⁺-ATPase. The major pool of GLAST/EAAT1, the only glutamate transporter subtype expressed by human fetal astrocytes in undifferentiated cultures, was restricted to the cytoplasmic compartment. Elevated glutamate concentrations (up to 50 μM) stimulated both glutamate uptake and Na⁺, K⁺-ATPase activity and concomitantly increased cell surface expression of GLAST and FXYD2/γ subunit of Na⁺, K⁺-ATPase. Intracellular accumulation of glutamate or its metabolites per se was not responsible for these changes since metabolically inert transport substrate, d-aspartate, exerted the same effect. Nanomolar concentrations of TFB-TBOA, a novel nontransportable inhibitor of glutamate carriers, almost completely reversed the action of glutamate or d-aspartate. In the same conditions (i.e. block of glutamate transport) monensin, a potent Na⁺ ionophore, had no significant effect neither on the activation of Na⁺, K⁺-ATPase nor on the cell surface expression of γ subunit or GLAST. In order to elucidate the roles of γ subunit in the glutamate uptake-dependent trafficking events or the activation of the astroglial sodium pump, in some cultures γ subunit/FXYD2 was effectively knocked down using siRNA silencing. Unlike the blocking effect of TFB-TBOA, the down-regulation of γ subunit had no effect neither on the trafficking nor activity of GLAST. However, the loss of γ subunit effectively abolished the glutamate uptake-dependent activation of Na⁺, K⁺-ATPase. Following withdrawal of siRNA from cultures, the expression levels of γ subunit and the sensitivity of Na⁺, K⁺-ATPase to glutamate/aspartate uptake have been concurrently restored. Thus, the activity of GLAST directs FXYD2 protein/γ subunit to the cell surface, that, in turn, leads to the activation of the astroglial sodium pump, presumably due to the modulatory effect of γ subunit on the kinetic parameters of catalytic α subunit(s) of Na⁺, K⁺-ATPase.

Introduction

Glutamic acid, a major excitatory amino acid in the brain, at higher concentrations can trigger aberrant metabolic pathways leading to neurodegeneration. The excess of extracellular glutamate is normally neutralized by high-affinity uptake mechanism executed by a family of glutamate transporter proteins: GLAST/EAAT1, GLT1/EAAT2, EAAC1/EAAT3, EAAT4 and EAAT5. The energy source for these secondary carriers is the electrochemical Na⁺ gradient maintained by Na⁺, K⁺-ATPase (Kanai and Hediger, 1992, Pines et al., 1992, Storck et al., 1992, Fairman et al., 1995, Arriza et al., 1997; for more references, see Gegelashvili and Schousboe, 1997, Gegelashvili and Schousboe, 1998, Gegelashvili et al., 2001, Danbolt, 2001).

In some pathological conditions (e.g. amyotrophic lateral sclerosis, ischemia, neurotrauma), this system fails to catch up with gradually increasing load of glutamate either because of the reduced expression of glutamate transporter proteins or due to the sustained alterations in ionic environment affecting kinetic properties of glutamate carriers. Moreover, as a result of severe cell energy deficit, glutamate transporters instead of protecting neurons, can aggravate excitotoxic insult by releasing intracellular glutamate through the reversed mode of operation (Choi, 1994, Rothstein et al., 1995, Rossi et al., 2000, Bonde et al., 2003a, for more references, see Yi and Hazell, 2006, Danbolt, 2001, Gegelashvili et al., 2001).

The major site for glutamate uptake in the brain is astroglia (Schousboe, 1981). Translocation of glutamate across the astroglial plasma membrane is executed by two distinct glutamate transporter subtypes, GLAST/EAAT1 and GLT1/EAAT2. Due to the emergence of the astroglial transport system as an obvious therapeutic target, molecular and cellular regulation of GLAST and GLT1 have been extensively studied in recent years (Gegelashvili et al., 1996, Gegelashvili et al., 1997, Gegelashvili et al., 2000, Swanson et al., 1996, Trotti et al., 1999, Zelenaia et al., 2000, Figiel et al., 2003, Rodriguez-Kern et al., 2003, Bonde et al., 2003b; see more references in Gegelashvili et al., 2001). Binding and subsequent translocation of glutamate into the cytosolic compartment by these two astroglial transporters serve both removal of the excess of glutamate from the perisynaptic space and nutritional supply of astrocytes. At the same time, elevated glutamate uptake dramatically increases energy demands of astrocytes, primarily due to ATP-consuming conversion of glutamate into glutamine as well as increased activity of Na⁺, K⁺-ATPase (Pellerin and Magistretti, 1994, Pellerin and Magistretti, 1997). The mechanism of glutamate uptake-dependent stimulation of the astroglial sodium pump has been explained by increased net influx of Na⁺ through glutamate transporter(s) (Magistretti and Chatton, 2005). Although it has been proposed that functional up-regulation of the catalytic α2 subunit of Na, K-ATPase is responsible for this effect (Pellerin and Magistretti, 1997), no experimental data on the roles of distinct molecular components of the sodium pump in this process have been provided so far. In the present study, we demonstrate that the activity of glutamate transporter GLAST/EAAT1 can effectively regulate cell surface expression of the γ subunit (FXYD2) of Na⁺, K⁺ ATPase in human fetal astrocytes.