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 alpha7 nicotinic acetylcholine (AcCho) receptor (alpha7 AcChoR) and its L248T mutated (mut) 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), mutalpha7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtalpha7 receptors decay much faster than those elicited by the mutalpha7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated alpha7 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 mutalpha7-GFP receptors, whereas those of the wtalpha7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mutalpha7-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 wtalpha7 and mutalpha7 receptors provides powerful tools to study the distribution and function of alpha7 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.
Palma
Eleonora
E
Istituto Pasteur-Fondazione Cenci Bolognetti and Dipartimento di Fisiologia Umana e Farmacologia, Universita' di Roma La Sapienza, P. le A. Moro 5, I00185 Roma, Italy.
Mileo
Anna M
AM
Martinez-Torres
Ataulfo
A
Eusebi
Fabrizio
F
Miledi
Ricardo
R
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D016764:Cell Polarity; D004594:Electrophysiology; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D006801:Humans; D015640:Ion Channel Gating; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009474:Neurons; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D021122:Protein Subunits; D021381:Protein Transport; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014982:Xenopus laevis; D064569:alpha7 Nicotinic Acetylcholine Receptor
D002038:Bungarotoxins; C579167:Chrna7 protein, human; D008164:Luminescent Proteins; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor; D049452:Green Fluorescent Proteins