Rapid recycling of glutamate transporters on the astroglial surface

Abstract

Glutamate uptake by astroglial transporters confines excitatory transmission to the synaptic cleft. The efficiency of this mechanism depends on the transporter dynamics in the astrocyte membrane, which remains poorly understood. Here, we visualise the main glial glutamate transporter GLT1 by generating its pH-sensitive fluorescent analogue, GLT1-SEP. FRAP-based imaging shows that 70-75% of GLT1-SEP dwell on the surface of rat brain astroglia, recycling with a lifetime of ~22 s. Genetic deletion of the C-terminus accelerates GLT1-SEP membrane turnover while disrupting its surface pattern, as revealed by single-molecule localisation microscopy. Excitatory activity boosts surface mobility of GLT1-SEP, involving its C-terminus, metabotropic glutamate receptors, intracellular Ca2+ and calcineurin-phosphatase activity, but not the broad-range kinase activity. The results suggest that membrane turnover, rather than lateral diffusion, is the main 'redeployment' route for the immobile fraction (20-30%) of surface-expressed GLT1. This finding reveals an important mechanism helping to control extrasynaptic escape of glutamate.

Estimating the glutamate transporter surface density in distinct sub-cellular compartments of mouse hippocampal astrocytes

“…Furthermore, we calculated the surface density of astrocytic membrane in stratum radiatum of the mouse hippocampus using 3D axial STEM tomography reconstructions (see below). Based on this analysis, the estimated astroglial surface density is 3.15 μm 2 /μm 3 . With these assumptions, we were able to estimate the average number of GLAST and GLT-1 molecules/μm 2 on the membrane surface of hippocampal astrocytes in tissue samples from different age groups: from 2 weeks to 21 month old mice.…”

“…Glutamate removal from the extracellular space relies on passive diffusion and active transport, a phenomenon mediated by transporter molecules abundantly expressed in the plasma membrane of astrocytes [1][2][3]. In the rat hippocampus, * 57% of excitatory synapses are contacted by astrocytic membranes, which surround * 43% of the synaptic perimeter [4].…”

“…Under basal conditions, the mobile GLT-1 molecules have a median instantaneous diffusion coefficient of 2.1 − 2.3 � 10 −2 μm 2 /s at 37˚C [2,45], which is similar to that of mobile GluN2B-containing NMDA receptors (2.5 � 10 −2 μm 2 /s [46]), but much higher than that of mobile AMPA (7.5 � 10 −3 μm 2 /s [47]) and GluN2A-containing NMDA receptors (7.5 � 10 −4 μm 2 /s [46]). The diffusion coefficient of GLT-1 is increased in astrocyte cell bodies and decreased upon glutamate exposure, suggesting that there is an activity-dependent confinement of GLT-1 around excitatory synapses [2,3,45]. In addition to being clustered (i.e., confined to hot spots), the distribution of glutamate transporters along the cell membrane appears to be highly non-uniform.…”

Super-resolution imaging to reveal the nanostructure of tripartite synapses

“…In a recent publication, we used fluorescence recovery after photobleaching in organotypic rat hippocampal sections to demonstrate that 70–75% of GLT-1 molecules dwell on the astrocyte surface, recycling with a lifetime of ∼22 s [ 117 ]. Using SMLM in cultured rat hippocampal cultures, we showed that GLT-1 surface expression and clustering behaviour relies on its C-terminus and its deletion accelerates GLT1 membrane turnover ( Figure 2 A) [ 117 ].…”

“… ( A ) Wide-field fluorescent images highlighting GLT-1 (green) and PSD95 (magenta) in mixed hippocampal astroglia-neuron cultures (top row). SMLM images showing individual labelled GLT-1 (green) and PSD95 (magenta) localisations (middle row depicting yellow squares in top row; bottom row depicting red squares in the middle row); modified from [ 117 ] with permission. ( B ) ExM image of dendrite and spines of a CA1 pyramidal neuron (green, YFP) and surrounding GLT-1 (red) labelling; with regions of ‘colocalisation’ highlighted in blue; modified from [ 119 ] with permission.…”