Trafficking of the Plasma Membrane γ-Aminobutyric Acid Transporter GAT1: SIZE AND RATES OF AN ACUTELY RECYCLING POOL

Dan Wang,Michael W Quick

doi:10.1074/jbc.m500381200

Abstract

Trafficking of the Plasma Membrane γ-Aminobutyric Acid Transporter GAT1: SIZE AND RATES OF AN ACUTELY RECYCLING POOL

Highlights

Plasma membrane neurotransmitter transporters rapidly traffic to and from the cell surface in neurons
Based upon estimates of GAT1 molecules in cortical boutons, the present data suggest that ϳ1000 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
To ensure that we could accurately assess the trafficking of GAT1, we verified the approach using the transferrin receptor (TfR)

Summary

SIZE AND RATES OF AN ACUTELY RECYCLING POOL*

Based upon estimates of GAT1 molecules in cortical boutons, the present data suggest that ϳ1000 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. In GAT1deficient mice generated by homologous recombinant gene targeting, the extracellular GABA concentration is chronically elevated due to the lack of uptake; such elevation increases tonic GABA receptor conductance and decreases phasic GABA inhibition [8] These findings suggest that GAT1 regulates the time course of GABAergic transmission. In the present experiments, modified biotinylation assays are applied to in vitro cortical neuron cultures to directly measure the basal trafficking rates of endogenous GAT1, to estimate the number of endogenous GAT1 molecules involved in acute trafficking, and to determine how three regulators of GAT1 trafficking exert their modulatory effects

MATERIALS AND METHODS

RESULTS

DISCUSSION