Some properties of human neuronal [alpha]7 nicotinic acetylcholine receptors fused to the green fluorescent protein

The functional properties and cellular localization of the human neuronal [alpha]7 nicotinic acetylcholine (AcCho) receptor ([alpha]7 AcChoR) and its L248T mutated (rout) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mut[alpha]7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wt[alpha]7 receptors decay much faster than those elicited by the mut[alpha]7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated [alpha]7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable 'run-down' of the AcCho currents generated by mut[alpha]7-GFP receptors, whereas those of the wtc[alpha]7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mut[alpha]7-GFP oocytes was accompanied by a marked decrease of [alpha]-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wt[alpha]7 and mut[alpha]7 receptors provides powerful tools to study the distribution and function of [alpha]7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins.