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. Fluorescence recovery after photobleaching-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.
Highlights
Excitatory transmission in the brain occurs mainly through the release of glutamate at chemical synapses
GLT1a was selected because it accounts for 90% of total GLT1 expression in astrocytes in the rat brain, with GLT1b and GLT1c accounting for ~6% and ~1%, respectively (Holmseth et al, 2009)
We used patch-clamp electrophysiology, immunostaining assays, and super-resolution single-molecule localisation microscopy (SMLM) imaging to confirm that glutamate transport properties of GLT1-Super-Ecliptic pHluorin (SEP) and its cell expression are generally compatible with its wild-type counterpart
Summary
Excitatory transmission in the brain occurs mainly through the release of glutamate at chemical synapses. Glutamate is taken up by high-affinity transporters that densely populate the plasma membrane of brain astrocytes (Wadiche et al, 1995a; Danbolt, 2001). Because synaptic vesicles release ~3000 glutamate molecules (Savtchenko et al, 2013) and because glutamate uptake cycle can take tens of milliseconds (Wadiche et al, 1995b), large numbers of transporter molecules have to be available near synapses to buffer the escaping glutamate (Lehre and Danbolt, 1998; Bergles et al, 2002). The high occurrence of GLT1 in astroglial plasma membranes (Danbolt, 2001) ensures that regular network activity does not overwhelm glutamate transport (Bergles and Jahr, 1998; Diamond and Jahr, 2000). The reduced availability of GLT1 in the synaptic environment has long been associated with pathologic conditions such as neurodegenerative diseases, stroke, or addiction (Maragakis and Rothstein, 2004; Fontana, 2015; Kruyer et al, 2019)
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