Ezrin mediates tethering of the gamma-aminobutyric acid transporter GAT1 to actin filaments via a C-terminal PDZ-interacting domain.
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.
Bioengineering Division, California Institute of Technology Pasadena, California 91125, USA.
P I
PI
Imoukhuede
Fraser J
FJ
Moss
Darren J
DJ
Michael
Robert H
RH
Chow
Henry A
HA
Lester
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D005786:Gene Expression Regulation; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D009068:Movement; D054731:PDZ Domains; D005680:gamma-Aminobutyric Acid / Q000378:metabolism; D000099149:Ezrin
D000199:Actins; D003598:Cytoskeletal Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D005680:gamma-Aminobutyric Acid; D000099149:Ezrin; C493414:Slc6a1 protein, mouse