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Ezrin Mediates Tethering of the γ-Aminobutyric Acid Transporter GAT1 to Actin Filaments Via a C-Terminal PDZ-Interacting Domain

Imoukhuede, P. I. and Moss, Fraser J. and Michael, Darren J. and Chow, Robert H. and Lester, Henry A. (2009) Ezrin Mediates Tethering of the γ-Aminobutyric Acid Transporter GAT1 to Actin Filaments Via a C-Terminal PDZ-Interacting Domain. Biophysical Journal, 96 (7). pp. 2949-2960. ISSN 0006-3495. PMCID PMC2711277. doi:10.1016/j.bpj.2008.11.070.

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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 (~50%) of membrane-localized GAT1 is immobile on the time scale investigated (~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 γ-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.

Item Type:Article
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URLURL TypeDescription CentralArticle
Moss, Fraser J.0000-0002-8519-6991
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2009 by the Biophysical Society. Received 3 July 2008; accepted 25 November 2008. Editor: Elliot L. Elson.. Available online 2 April 2009. We thank Michael Quick and Elaine Bearer for valuable discussions. This research was supported by grants from the National Institutes of Health (DA-09121, DK-60623, and NS-11756), by an American Heart Association Postdoctoral Fellowship to F.M., and by the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology.
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American Heart AssociationUNSPECIFIED
Millard and Muriel Jacobs Genetics and Genomics LaboratoryUNSPECIFIED
Issue or Number:7
PubMed Central ID:PMC2711277
Record Number:CaltechAUTHORS:20090903-090332845
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Official Citation:Ezrin Mediates Tethering of the γ-Aminobutyric Acid Transporter GAT1 to Actin Filaments Via a C-Terminal PDZ-Interacting Domain. Imoukhuede, P.I.; Moss, Fraser J.; Michael, Darren J.; Chow, Robert H.; Lester, Henry A. doi:10.1016/j.bpj.2008.11.070 (volume 96 issue 7 pp.2949 - 2960)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:15562
Deposited By: Ruth Sustaita
Deposited On:14 Sep 2009 21:00
Last Modified:08 Nov 2021 23:20

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