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Neurotransmitter transporters function in synaptic signaling in part through the sequestration and removal of neurotransmitter from the synaptic cleft. A recurring theme of transporters is that many can be functionally regulated by protein kinase C (PKC); some of this regulation occurs via a redistribution of the transporter protein between the plasma membrane and the cytoplasm. The endogenous triggers that lead to PKC-mediated transporter redistribution have not been elucidated. G-protein-coupled receptors that activate PKC are likely candidates to initiate transporter redistribution. We tested this hypothesis by examining the rat brain GABA transporter GAT1 endogenously expressed in hippocampal neurons. Specific agonists of G-protein-coupled acetylcholine, glutamate, and serotonin receptors downregulate GAT1 function. This functional inhibition is dose-dependent, mimicked by PKC activators, and prevented by specific receptor antagonists and PKC inhibitors. Surface biotinylation experiments show that the receptor-mediated functional inhibition correlates with a redistribution of GAT1 from the plasma membrane to intracellular locations. These data demonstrate (1) that endogenous GAT1 function can be regulated by PKC via subcellular redistribution, and (2) that signaling via several different G-protein-coupled receptors can mediate this effect. These results raise the possibility that some effects of G-protein-mediated alterations in synaptic signaling might occur through changes in the number of transporters expressed on the plasma membrane and subsequent effects on synaptic neurotransmitter levels.

gamma-Aminobutyric acid (GABA) transporters on neurons and glia at or near the synapse function to remove GABA from the synaptic cleft. Recent evidence suggests that GABA transporter function can be regulated, although the initial triggers for such regulation are not known. One hypothesis is that transporter function is modulated by extracellular GABA concentration, thus providing a feedback mechanism for the control of neurotransmitter levels at the synapse. To test this hypothesis, GABA uptake assays were performed on primary dissociated rat hippocampal cultures that endogenously express GABA transporters and on mammalian cells stably expressing the cloned rat brain GABA transporter GAT1. In both experimental systems, extracellular GABA induces chronic changes in GABA transport that occur in a dose-dependent and time-dependent manner. In addition to GABA, ACHC and nipecotic acid, both substrates of GAT1, up-regulate transport; GAT1 transport inhibitors that are not transporter substrates down-regulate transport. These changes occur in the presence of blockers of both GABAA and GABAB receptors, occur in the presence of protein synthesis inhibitors, and are not influenced by intracellular GABA. Surface biotinylation experiments reveal that the increase in transport is correlated with an increase in surface transporter expression. This increase in surface expression is due, at least in part, to a slowing of GAT1 internalization in the presence of extracellular GABA. These data suggest that the GABA transporter fine-tunes its function in response to extracellular GABA and would act to maintain a constant level of neurotransmitter at the synaptic cleft.

Syntaxin 1A inhibits GABA uptake of an endogenous GABA transporter in neuronal cultures from rat hippocampus and in reconstitution systems expressing the cloned rat brain GABA transporter GAT1. Evidence of interactions between syntaxin 1A and GAT1 comes from three experimental approaches: botulinum toxin cleavage of syntaxin 1A, syntaxin 1A antisense treatments, and coimmunoprecipitation of a complex containing GAT1 and syntaxin 1A. Protein kinase C (PKC), shown previously to modulate GABA transporter function, exerts its modulatory effects by regulating the availability of syntaxin 1A to interact with the transporter, and a transporter mutant that fails to interact with syntaxin 1A is not regulated by PKC. These results suggest a new target for regulation by syntaxin 1A and a novel mechanism for controlling the machinery involved in both neurotransmitter release and reuptake.

The mutations responsible for several human neurodegenerative disorders are expansions of translated CAG repeats beyond a normal size range. To address the role of repeat context, we have introduced a 146-unit CAG repeat into the mouse hypoxanthine phosphoribosyltransferase gene (Hprt). Mutant mice express a form of the HPRT protein that contains a long polyglutamine repeat. These mice develop a phenotype similar to the human translated CAG repeat disorders. Repeat containing mice show a late onset neurological phenotype that progresses to premature death. Neuronal intranuclear inclusions are present in affected mice. Our results show that CAG repeats do not need to be located within one of the classic repeat disorder genes to have a neurotoxic effect.