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GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET

Moss, Fraser J. and Imoukhuede, P. I. and Scott, Kimberly and Hu, Jia and Jankowsky, Joanna L. and Quick, Michael W. and Lester, Henry A. (2009) GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET. Journal of General Physiology, 134 (6). pp. 489-521. ISSN 0022-1295. PMCID PMC2806419. doi:10.1085/jgp.200910314.

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The mouse γ-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [^3H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [^3H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ~30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst.

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 Moss et al. This article is distributed under the terms of an Attribution–Noncommercial– Share Alike–No Mirror Sites license for the first six months after the publication date (see After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at Submitted: 7 August 2009. Accepted: 2 November 2009. Edward N. Pugh Jr. served as editor. We thank Robert Chow, Cagdas Son, Rigo Pantoja, and Rahul Srinivasan for discussion. We thank Jo Ann Trinkle and Elisha Mackey for technical assistance. We also acknowledge Ryan Morin of Michael Smith Genome Sciences Centre, BC Cancer Agency Vancouver, BC, Canada for his assistance with the bioinformatics. This research is supported by grants from the National Institutes of Health (grants DA-09121, DA-10509, and NS-11756). F.J. Moss received an American Heart Association Postdoctoral Fellowship. Some experiments used the facilities of the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology.
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American Heart AssociationUNSPECIFIED
Issue or Number:6
PubMed Central ID:PMC2806419
Record Number:CaltechAUTHORS:20091218-122259894
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Official Citation:Fraser J. Moss, P.I. Imoukhuede, Kimberly Scott, Jia Hu, Joanna L. Jankowsky, Michael W. Quick, and Henry A. Lester GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET J. Gen. Physiol. 134: 489-521; published online before print as 10.1085/jgp.200910314
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:17002
Deposited By: Tony Diaz
Deposited On:06 Jan 2010 00:09
Last Modified:08 Nov 2021 23:31

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