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

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

Ezrin Mediates Tethering of the γ-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 (~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