Trafficking of the plasma membrane gamma-aminobutyric acid transporter GAT1. Size and rates of an acutely recycling pool

Plasma membrane neurotransmitter transporters rapidly traffic to and from the cell surface in neurons. This trafficking may be important in regulating neuronal signaling. Such regulation will be subject to the number of trafficking transporters and their trafficking rates. In the present study, we define an acutely recycling pool of endogenous gamma-aminobutyric acid transporters (GAT1) in cortical neurons that comprises approximately one-third of total cellular GAT1. Kinetic analysis of this pool estimates exocytosis and endocytosis time constants of 1.6 and 0.9 min, respectively, and thus approximately one-third of the recycling pool is plasma membrane resident in the basal state. Recent evidence shows that GAT1 substrates, second messengers, and interacting proteins regulate GAT1 trafficking. These triggers could act by altering trafficking rates or by changing the recycling pool size. In the present study we examine three GAT1 modulators. Calcium depletion decreases GAT1 surface expression by diminishing the recycling pool size. Sucrose increases GAT1 surface expression by blocking clathrin- and dynamin-dependent endocytosis, but it does not change the recycling pool size. Protein kinase C decreases surface GAT1 expression by increasing the endocytosis rate, but it does not change the exocytosis rate or the recycling pool size. Based upon estimates of GAT1 molecules in cortical boutons, the present data suggest that approximately 1000 transporters comprise the acutely recycling pool, of which 300 are on the surface in the basal state, and five transporters insert into the plasma membrane every second. This insertion could represent the fusion of one transporter-containing vesicle.