Nicotinic Acetylcholine Receptor Î±7 Subunits With A C2 Cytoplasmic Loop Yellow Fluorescent Protein Insertion Form Functional Receptors
Aim: Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR Î±7 subunit without compromising formation of functional receptors. Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR Î±7 subunit (Î±7Y). SH-EP1 cells were transfected with mouse nAChR wild type Î±7 subunits (Î±7) or with Î±7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125 I-labeled Î±-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy. Results: Whole-cell currents revealed that Î±7Y nAChRs and Î±7 nAChRs were functional with comparable EC 50 values for the Î±7 nAChR-selective agonist, choline, and IC 50 values for the Î±7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that Î±7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little Î±7Y localized to the plasma membrane, typical of Î±7 nAChRs. Conclusion: nAChRs composed as homooligomers of Î±7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of Î±7 nAChRs. Î±7Y nAChRs may be used to elucidate properties of Î±7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion. Â© 2009 CPS and SIMM.
Acta Pharmacologica Sinica
Digital Object Identifier (DOI)
Murray, Teresa A.; Liu, Qiang; Whiteaker, Paul; Wu, Jie; and Lukas, Ronald J., "Nicotinic Acetylcholine Receptor Î±7 Subunits With A C2 Cytoplasmic Loop Yellow Fluorescent Protein Insertion Form Functional Receptors" (2009). Neurobiology. 457.