Quantification Of Sensitized FRET From Fluorescent GAT1 γ-aminobutyric Acid Transporters Distinguishes Between Subsurface And Plasma Membrane Resident Oligomers And Predicts Function

γ-aminobutyric acid transporter (mGAT1) oligomerization was studied in subcellular regions using pixel-by-pixel analysis of normalized Forster resonance energy transfer (NFRET) images. Nineteen fluorescent mGAT1 protein designs were functionally characterized with non-saturated, linear uptake assays in N2a cells. Some constructs were functionally non-distinguishable from wild-type mGAT1; others oligomerized but did not traffic correctly; and some showed deficits in both assembly and trafficking. For Fluorescent mGAT1s that possess >75% wild-type function, NFRET in the peripheral region of interest (ROI) was ≥ 140% of the value in the perinuclear ROI. NFRET amplitude distributions of pixels from wild-type functioning constructs were best fit to three Gaussians. In the peripheral ROI, the highest-NFRET component comprised ∼30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool expected to be inserted into the plasma membrane in the basal state. The high-NFRET component was absent in intracellular or peripheral ROIs of cells expressing fluorescent mGAT1 constructs with ≤65% wild-type function. Thus, pixel-by-pixel analysis of NFRET quantitatively distinguishes between subsurface and membrane-inserted transporter complexes; and mGAT1 exhibits up to three quantifiable oligomerization states. Support: NIH (DA-09121; DA-10509; NS-11756), AHA (postdoctoral fellowship to FJM).