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Abstract
A high density of neurotransmitter transporters on axons and presynaptic boutons is
required for the efficient clearance of neurotransmitters from the synapse. Therefore,
regulators of transporter trafficking (insertion, retrieval, and confinement) can
play an important role in maintaining the transporter density necessary for effective
function. We determined the interactions that confine GAT1 at the membrane by investigating
the lateral mobility of GAT1-yellow fluorescent protein-8 (YFP8) expressed in neuroblastoma
2a cells. Through fluorescence recovery after photobleaching, we found that a significant
fraction ( approximately 50%) of membrane-localized GAT1 is immobile on the time scale
investigated ( approximately 150 s). The mobility of the transporter can be increased
by depolymerizing actin or by interrupting the GAT1 postsynaptic density 95/Discs
large/zona occludens 1 (PDZ)-interacting domain. Microtubule depolymerization, in
contrast, does not affect GAT1 membrane mobility. We also identified ezrin as a major
GAT1 adaptor to actin. Förster resonance energy transfer suggests that GAT1-YFP8 and
cyan fluorescent (CFP) tagged ezrin (ezrin-CFP) exist within a complex that has a
Förster resonance energy transfer efficiency of 19% +/- 2%. This interaction can be
diminished by disrupting the actin cytoskeleton. In addition, the disruption of actin
results in a >3-fold increase in gamma-aminobutyric acid uptake, apparently via a
mechanism distinct from the PDZ-interacting protein. Our data reveal that actin confines
GAT1 to the plasma membrane via ezrin, and this interaction is mediated through the
PDZ-interacting domain of GAT1.