Abstract
Astrocytes are glial cells that have an intimate physical and functional association with synapses in the brain. One of their main roles is to recycle the neurotransmitters glutamate and gamma-aminobutyric acid (GABA), as a component of the glutamate/GABA-glutamine cycle. They perform this function by sequestering neurotransmitters and releasing glutamine via the neutral amino acid transporter SNAT3. In this way, astrocytes regulate the availability of neurotransmitters and subsequently influence synaptic function. Since many plasma membrane transporters are regulated by protein kinase C (PKC), the aim of this study was to understand how PKC influences SNAT3 glutamine transport in astrocytes located immediately adjacent to synapses. We studied SNAT3 transport by whole-cell patch-clamping and fluorescence pH imaging of single astrocytes in acutely isolated brainstem slices, adjacent to the calyx of the Held synapse. Activation of SNAT3-mediated glutamine transport in these astrocytes was reduced to 77 ± 6% when PKC was activated with phorbol 12-myristate 13-acetate (PMA). This effect was very rapid (within ~20 min) and eliminated by application of bisindolylmaleimide I (Bis I) or 7-hydroxystaurosporine (UCN-01), suggesting that activation of conventional isoforms of PKC reduces SNAT3 function. In addition, cell surface biotinylation experiments in these brain slices show that the amount of SNAT3 in the plasma membrane is reduced by a comparable amount (to 68 ± 5%) upon activation of PKC. This indicates a role for PKC in dynamically controlling the trafficking of SNAT3 transporters in astrocytes in situ. These data demonstrate that PKC rapidly regulates the astrocytic glutamine release mechanism, which would influence the glutamine availability for adjacent synapses and control levels of neurotransmission.
Highlights
Throughout the body, the neutral amino acid glutamine plays a central role in tissue nitrogen homeostasis and intercellular nutrition [1]
We have studied SNAT3 transporter function in astrocytes located immediately adjacent to the calyx of Held synapse in brain slices from the auditory brainstem of rats and mice
Astrocytes were positively identified by the morphology visible under fluorescent illumination, showing a characteristic branching structure and close association with the calyx of Held synapse around the medial nucleus of the trapezoid body (MNTB) cell soma (Figure 1b)
Summary
Throughout the body, the neutral amino acid glutamine plays a central role in tissue nitrogen homeostasis and intercellular nutrition [1]. The principal excitatory neurotransmitter glutamate is released to activate postsynaptic cells and is subsequently sequestered into neighbouring astrocytes to terminate the neurotransmitter signal [2]. In these astrocytes, glutamate is amidated to form glutamine, which is transported out of astrocytes and back into neurons for hydrolysis back to glutamate, forming the glutamate–glutamine cycle (see reviews by [3,4,5,6]). As the brain’s main inhibitory neurotransmitter GABA is synthesised from glutamate, glutamine shuttling from astrocytes to neurons is important for the maintenance of inhibitory neurotransmission [12,13,14,15]
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