Plasma membrane GABA transporters reside on distinct vesicles and undergo rapid regulated recycling

Plasma membrane neurotransmitter transporters affect synaptic signaling through transmitter sequestration. Transporters redistribute to and from the plasma membrane, suggesting a role for trafficking in regulating synaptic transmitter levels. One method for controlling transmitter levels would be to regulate transporter redistribution in parallel with transmitter release. Thus, how similar are these processes? We show that the trafficking of the GABA transporter GAT1 resembles the trafficking of neurotransmitter-filled synaptic vesicles: (1) transporters located on the plasma membrane are internalized and reinserted into the plasma membrane on the order of minutes; (2) the rate of recycling is depolarization and calcium dependent; (3) GAT1 internalization is associated with clathrin and dynamin; and (4) intracellular GAT1 is associated with multiple compartments and, more importantly, is found on a distinct class of vesicles. These vesicles are clear, ∼50 nm in diameter, and contain many proteins found on neurotransmitter-containing small synaptic vesicles; however, they appear to lack several traditional small synaptic vesicle proteins, such as synaptophysin and the vesicular GABA transporter. These data provide additional support for the hypothesis that GABA transporters traffic in parallel with neurotransmitter- containing small synaptic vesicles and also raise the possibility that some fraction of vesicles found in GABAergic neurons may not be participating in transmitter release but rather in the rapid regulated redistribution of membrane proteins involved in transmitter uptake.