Ric-3 chaperone-mediated stable cell-surface expression of the neuronal alpha7 nicotinic acetylcholine receptor in mammalian cells.
OBJECTIVE
Studies of the alpha7-type neuronal nicotinic acetylcholine receptor (AChR), one of the receptor forms involved in many physiologically relevant processes in the central nervous system, have been hampered by the inability of this homomeric protein to assemble in most heterologous expression systems. In a recent study, it was shown that the chaperone Ric-3 is necessary for the maturation and functional expression of alpha7-type AChRs(1). The current work aims at obtaining and characterizing a cell line with high functional expression of the human alpha7 AChR.


METHODS
Ric-3 cDNA was incorporated into SHE-P1-halpha7 cells expressing the alpha7-type AChR. Functional studies were undertaken using single-channel patch-clamp recordings. Equilibrium and kinetic [(125)I]alpha-bungarotoxin binding assays, as well as fluorescence microscopy using fluorescent alpha-bungarotoxin, anti-alpha7 antibody, and GFP-alpha7 were performed on the new clone.


RESULTS
The human alpha7-type AChR was stably expressed in a new cell line, which we coined SHE-P1-halpha7-Ric-3, by co-expression of the chaperone Ric-3. Cell-surface AChRs exhibited [(125)I]alphaBTX saturable binding with an apparent K(D) of about 55 nmol/L. Fluorescence microscopy revealed dispersed and micro-clustered AChR aggregates at the surface of SHE-P1-halpha7-Ric-3 cells. Larger micron-sized clusters were observed in the absence of receptor-clustering proteins or upon aggregation with anti-alpha7 antibodies. In contrast, chaperone-less SHE-P1-halpha7 cells expressed only intracellular alpha7 AChRs and failed to produce detectable single-channel currents.


CONCLUSIONS
The production of a stable and functional cell line of neuroepithelial lineage with robust cell-surface expression of neuronal alpha7-type AChR, as reported here, constitutes an important advance in the study of homomeric receptors in mammalian cells.
Vallés
Ana Sofía
AS
Instituto de Investigaciones Bioquímicas de Bahía Blanca and UNESCO Chair of Biophysics and Molecular Neurobiology, CC 857, B8000FWB, Bahía Blanca, Argentina.
Roccamo
Ana M
AM
Barrantes
Francisco J
FJ
D002038:Bungarotoxins / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane; D018076:DNA, Complementary / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011978:Receptors, Nicotinic / Q000378:metabolism*; D064569:alpha7 Nicotinic Acetylcholine Receptor
D002038:Bungarotoxins; C579167:Chrna7 protein, human; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; C496701:RIC3 protein, human; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor