Activity dependent internalization of the glutamate transporter GLT-1 requires calcium entry through the NCX sodium/calcium exchanger

Abstract

GLT-1 is the main glutamate transporter in the brain and its trafficking controls its availability at the cell surface, thereby shaping glutamatergic neurotransmission under physiological and pathological conditions. Extracellular glutamate is known to trigger ubiquitin-dependent GLT-1 internalization from the surface of the cell to the intracellular compartment, yet here we show that internalization also requires the participation of calcium ions. Consistent with previous studies, the addition of glutamate (1 mM) to mixed primary cultures (containing neurons and astrocytes) promotes GLT-1 internalization, an effect that was suppressed in the absence of extracellular Ca. The pathways of Ca mobilization by astrocytes were analyzed in these mixed cultures using the genetically encoded calcium sensor GCaMP6f. A complex pattern of calcium entry was activated by glutamate, with a dramatic and rapid rise in the intracellular Ca concentration partially driven by glutamate transporters, especially in the initial stages after exposure to glutamate. The Na/Ca exchanger (NCX) plays a dominant role in this Ca mobilization and its blockade suppresses the glutamate induced internalization of GLT-1, both in astrocytes and in a more straightforward experimental system like HEK293 cells transiently transfected with GLT-1. This regulatory mechanism might be relevant to control the amount of GLT-1 transporter at the cell surface in conditions like ischemia or traumatic brain injury, where extracellular concentrations of glutamate are persistently elevated and they promote rapid Ca mobilization.

Sodium-Calcium Exchanger Mediates Sensory-Evoked Glial Calcium Transients in the Developing Retinotectal System

“…Glial calcium responses in our experiments often desensitized to the visual stimulus within the first couple of minutes of repeated stimulation, and consistent with the mechanism we have proposed above, it has been observed elsewhere that the activity-dependent internalization of the glial glutamate transporter GLT-1 requires calcium entry through NCX 45 . This suggests that visual stimulation may lead to the internalization of EAATs here as well.…”

“…has been shown in zebrafish12 . It will be valuable to mechanistically dissect glia to neuron signals occurring in the retinotectal circuit and determine how they modulate visual processing.The temporal kinetics of the calcium responses occurring during visual stimulation in these radial astrocytes match closely with those observed in astrocytes in the visual cortex of adult ferrets15 and more recently in the somatosensory cortex of adult mice44 , where sensory-evoked Glial calcium responses in our experiments often desensitized to the visual stimulus within the first couple of minutes of repeated stimulation, and consistent with the mechanism we have proposed above, it has been observed elsewhere that the activity-dependent internalization of the glial glutamate transporter GLT-1 requires calcium entry through NCX45 . This suggests that visual stimulation may lead to the internalization of EAATs here as well.…”

Identification by proximity labeling of novel lipidic and proteinaceous potential partners of the dopamine transporter

“…Primary brain cortex cultures from 18-day-old rat fetuses were established as described elsewhere [ 51 ], isolating the tissue in Hanks balanced salt solution (Invitrogen), and dissociating the cells with 0.25% trypsin and 4 mg/ml DNase. The cells were incubated for 4 h in plating buffer (DMEM containing 10% FCS and supplemented with 10 mM glucose, 10 mM sodium pyruvate, 0.5 mM glutamine, 0.05 mg/ml gentamicin, 0.01% streptomycin, 100 µU/ml penicillin G) and the buffer was then replaced by culture medium (Neurobasal/B27 50:1 by volume, containing 0.5 mM glutamine).…”

“…Primary cultures of neurons (transfected or not) or transfected cell lines were fixed in 4% paraformaldehyde/PBS, blocked, permeabilized, and incubated with primary and secondary antibodies as described previously [ 51 ]. The coverslips were mounted in fluoromount-G for analysis.…”

The Regulation of Astrocytic Glutamate Transporters in Health and Neurodegenerative Diseases

“…Indeed, immobilising the diffusion of EAAT2 was found to alter synaptic kinetics, increasing both the rise and decay time of spontaneous excitatory postsynaptic currents, although total glutamate clearance was unaltered [51]. As well as surface diffusion, EAATs also undergo surface trafficking, with glutamate triggering Ca + -dependent internalization of EAAT2 through endocytosis [116,117]. On the other hand, surface expression of EAAT1 has been shown to be increased by glutamate application, as well as insulin-like growth factor through phosphatidylinositol-3-kinase signalling, whereas the ubiquitin ligase neuronal developmentally downregulated gene 4, isoform 2 (Nedd4-2) has been shown to decrease EAAT1 surface expression [115,118,119].…”