Abstract
Stargazin is an accessory protein of AMPA receptors that enhances surface expression and also affects the biophysical properties of the receptor. AMPA receptor domains necessary for either of these two processes have not yet been identified. Here, we used confocal imaging and electrophysiology of heterologously expressed, fluorophore-tagged GluR1, GluR2, and stargazin to study surface expression and desensitization kinetics. Stargazin-mediated trafficking was sensitive to the nature of the AMPA receptor cytoplasmic domain. The insertion of YFP after residue 15 of the truncated cytoplasmic tail of GluR1i perturbed stargazin-mediated trafficking of the receptor but not its modulation of desensitization kinetics. This construct also failed to permit fluorescence resonance energy transfer (FRET) with stargazin in the endoplasmic reticulum (ER), whereas FRET between fluorophore-tagged stargazin and non-truncated AMPA receptors demonstrated a specific interaction between these proteins, both in the ER and the plasma membrane. Rather than encoding a specific binding site, the fluorophore-tagged C terminus may restrict access to one or more ER retention sites. Although perturbations of the C terminus impeded stargazin-mediated trafficking to the plasma membrane, the effects of stargazin on the biophysical properties of AMPA receptors (i.e. modulation of desensitization) remained intact. These data provide strong evidence that the AMPA receptor domains required for stargazin modulation of gating and trafficking are separable.
Highlights
Stargazin Alters GluR1i Distribution in the Cytosol and the Plasma Membrane—To assay stargazin-mediated trafficking of AMPA receptors, in-frame chimeric proteins were constructed by fusing a fluorescent protein (ECFP or EYFP) to the C-terminal domain of either GluR1 (R1) or GluR2 (R2)
Confocal imaging through live cells that expressed fluorophore-tagged AMPA receptors allowed us to distinguish between accumulation of protein in reticular, perinuclear networks or within the plasma membrane, such that the labeled protein formed prominent surface rings
When R1i81YFP was cotransfected with stargazin, the plasma membrane intensity relative to the cytosolic intensity was markedly increased in a population of cells (Fig. 1b and Table 1), because of decreased cytosolic expression of R1i81YFP
Summary
Transient Transfections for Electrophysiology—Human embryonic kidney 293 (HEK-293) fibroblasts (CRL 1573; American Type Culture Collection) were cultured as described previously [25]. Confocal Imaging of GFP and Fluorescent Microspheres— Cells transfected with stgGFP were excited using the 488-nm line of an argon laser attenuated to 6%. Immunofluorescence for Confocal Imaging—After transfection as described above for 3 days with 0.3 g of FLAG-GluR4i cDNA with or without 0.6 g of stg, cells were rinsed with phosphate-buffered saline fixed with 4% paraformaldehyde for 15–20 min. Cells were again washed 3 ϫ 10 min in blocking solution rinsed several times in phosphate-buffered saline before imaging with an Olympus FV1000 confocal microscope. Quantitation of Surface Expression—Transfected HEK-293 cells were visualized using a confocal microscope and divided into 3 categories (where a “ring” is defined as markedly enhanced surface expression, which if imaged as an optical slice with a confocal microscope, appears as enhanced fluorescent intensity co-localizing with the plasma membrane): definitely rings, definitely no rings, and unclassifiable.
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