GABA is the major inhibitory
neurotransmitter in the CNS and changes in GABAergic neurotransmission affect the overall activity of neuronal networks. The uptake of
GABA into synaptic vesicles is mediated by the
vesicular GABA transporter (VGAT), and changes in the expression of the transporter directly regulate
neurotransmitter release. In this work we investigated the changes in VGAT
protein levels during
ischemia and in excitotoxic conditions, which may affect the demise process. We found that VGAT is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with
glutamate, giving rise to a stable truncated cleavage product (tVGAT). VGAT cleavage was also observed after transient
middle cerebral artery occlusion in mice, a
cerebral ischemia model, and following intrahippocampal injection of
kainate, but no effect was observed in transgenic mice overexpressing
calpastatin, a
calpain inhibitor. Incubation of isolated cerebrocortical synaptic vesicles with recombinant
calpain also induced the cleavage of VGAT and formation of stable tVGAT. Immunoblot experiments using
antibodies targeting different regions of VGAT and N-terminal sequencing analysis showed that
calpain cleaves the transporter in the N-terminal region, at
amino acids 52 and 60. Immunocytochemistry of GABAergic striatal neurons expressing GFP fusion
proteins with the full-length VGAT or tVGAT showed that cleavage of the transporter induces a loss of synaptic delivery, leading to a homogeneous distribution of the
protein along neurites. Our results show that excitotoxicity downregulates full-length VGAT, with a concomitant generation of tVGAT, which is likely to affect GABAergic neurotransmission and may influence cell death during
ischemia.