Regions of α-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid receptor subunits that are permissive for the insertion of green fluorescent protein

• Published in: Neuroscience Vol. 141; no. 2; pp. 837 - 849
• Main Authors: Sheridan, D.L., Robert, A., Cho, C.H., Howe, J.R., Hughes, T.E.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01-01-2006; Elsevier
• Subjects: Amino Acid Sequence; AMPA receptor; Biological and medical sciences; Blotting, Western - methods; Cell Line; Flow Cytometry - methods; Fundamental and applied biological sciences. Psychology; fusion protein; Gene Expression - physiology; glutamate receptor subunit; Glutamic Acid - pharmacology; green fluorescent protein; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Humans; Immunohistochemistry - methods; insertional tagging; Membrane Potentials - drug effects; Membrane Potentials - genetics; Membrane Potentials - physiology; Mutagenesis - physiology; Oligopeptides; Patch-Clamp Techniques - methods; Peptides - genetics; Peptides - metabolism; Protein Subunits - genetics; Protein Subunits - metabolism; Receptors, AMPA - chemistry; Receptors, AMPA - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Tn 5 transposon; Transfection - methods; Vertebrates: nervous system and sense organs; YFP; AMPA; GluR; glutamate receptor subunit; CFP; green fluorescent protein; mGluR; GFP; ATD; AMPA receptor; fusion protein; CTD; insertional tagging; ME; Tn 5 transposon; FRET; Green fluorescent protein; Glutamate receptor; Subunit; Tn5 transposon; Biological receptor
• DOI: 10.1016/j.neuroscience.2006.04.052
• EISSN: 1873-7544
• ISSN: 0306-4522

The green fluorescent protein can be fused to the ends of a mature glutamate receptor subunit to produce functional, fluorescent receptors. However, there are good reasons to search for internal regions of receptor subunits that can tolerate green fluorescent protein insertion. First, internal insertions of green fluorescent protein may produce functional, fluorescent subunits that traffic more correctly. Second, fluorescent proteins inserted near interacting surfaces of subunits could potentially create reagents suitable for fluorescence resonance energy transfer measurements. Finally, internal green fluorescent protein insertions could potentially produce subunits capable of signaling conformational changes through intrinsic changes in fluorescence intensity. To identify regions of receptor subunits that are permissive for green fluorescent protein insertion, we used a series of recombinant transposons to create fluorescent protein insertions in three α-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid receptor subunits. A combined analysis of the relative fluorescence intensity and glutamate-gated ion channel function of 69 different green fluorescent protein fusion proteins identified permissive zones for the creation of bright and fully functional receptor subunits in the C-terminal portion of the amino terminal domain, the intracellular tail of the carboxy terminal domain, and within the pore-forming regions of the channel.