Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

Abstract

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 alpha7 subunit without compromising formation of functional receptors. A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplasmic loop between the third and fourth transmembrane domains of the mouse nAChR alpha7 subunit (alpha7Y). SH-EP1 cells were transfected with mouse nAChR wild type alpha7 subunits (alpha7) or with alpha7Y 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 alpha-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy. Whole-cell currents revealed that alpha7Y nAChRs and alpha7 nAChRs were functional with comparable EC(50) values for the alpha7 nAChR-selective agonist, choline, and IC(50) values for the alpha7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that alpha7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little alpha7Y localized to the plasma membrane, typical of alpha7 nAChRs. nAChRs composed as homooligomers of alpha7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of alpha7 nAChRs. alpha7Y nAChRs may be used to elucidate properties of alpha7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.