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

Abstract

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.

Transport Mechanisms of Nucleosides and Nucleoside Analogues ReverseTranscriptase Inhibitors in the Brain

“…hENT1 was then trafficked to the plasma membrane in association with the microtubule network in a variety of vesicles; in the plasma membrane hENT1 was co-localized with actin, which suggested that this transporter was anchored in the membrane by the actin cytoskeleton (Nivillac et al, 2011). This finding was consistent with previous observations for some other transporters, such as mouse GABA transporter 1 (Moss et al, 2009). After about an hour at the plasma membrane hENT1 internalized and interestingly only a proportion of the transporter population was recycled, which could indicate an efficient mechanism for fine-tuning, similar to what was described for the organic anion exchanger OAT1 .…”

Lynx1 Shifts α4β2 Nicotinic Receptor Subunit Stoichiometry by Affecting Assembly in the Endoplasmic Reticulum

“…Confocal Microscopy-N2a cells and neurons were imaged as described previously (19,20) in L15 buffer (150 mM NaCl, 4 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 10 mM HEPES, and 10 mM D-glucose, pH 7.4), a CO 2 -independent medium. Briefly, cells were imaged with a Nikon (Nikon Instruments, Melville, NY) C1 laser-scanning confocal microscope system equipped with spectral imaging capabilities and a Prior (Rockland, ME) remote focus device, and a Nikon Plan Apo 60ϫ 1.40 NA oil objective.…”

The Duplicated α7 Subunits Assemble and Form Functional Nicotinic Receptors with the Full-length α7

“…The binding fraction, the fraction of donor that interacts with the acceptor, and FRET efficiency were calculated as described under "Experimental Procedures." In the context of analyses presented elsewhere, we believe that the binding fraction is best interpreted as the fraction of donors adjacent to one or more acceptors (23,28,31,32). These two metrics are relatively insensitive to fluorophore concentration and light-path length, conditions that are poorly controlled inside a cell (35,36).…”

“…When the fluorescent proteins are fused into the M3-M4 loop of nicotinic receptor subunits, subunits within pentamers undergo FRET. We have previously developed experimental approaches, well supported by theory, to analyze the assembly and subunit stoichiometry of such subunits within Cys loop receptors (23,28,(31)(32)(33). For the present experiments, important guidelines arise from the strong distance dependence of FRET.…”