Mutational analysis of roles for extracellular cysteine residues in the assembly and function of human alpha 7-nicotinic acetylcholine receptors.
Nicotinic acetylcholine receptors (nAChR) containing alpha7 subunits self-assemble into simple, homopentameric complexes. However, successful heterologous expression of functional alpha7-nAChR has only been achieved in a few host cell types, such as the SH-EP1 human epithelial cell line. All ionotropic glycine receptor, GABA(A) receptor, 5-HT(3) receptor, and nAChR subunits contain a pair of highly conserved cysteine residues (C150 and C164 for alpha7 subunits) in their N-terminal extracellular domain. These residues are thought to be involved in the formation of a conserved cystine loop that is critical to the proper folding and assembly of subunits. However, nAChR alpha7 (and alpha8) subunits also contain a third cysteine residue, C138, N-terminal to the conserved cysteine pair. Using SH-EP1 cells as a host for heterologous expression, we evaluated the roles of C138, C150, and C164 in subunit folding, assembly, and cell surface expression and function of alpha7-nAChR. Results indicate that mutation of C138, but not of C150 or C164, yields an nAChR that can assemble to form (125)I-labeled alpha-bungarotoxin binding sites expressed on the cell surface. Further, whole-cell patch clamp recordings demonstrate that mutation of C138 to alanine does not alter the function of the fully assembled alpha7-nAChR. These results indicate that C150 and C164 are required for surface expression, but that C138 is neither necessary for nor inhibitory toward the surface expression and function of human alpha7-nAChR. These results suggest that disulfide bond formation between C138 and either C150 or C164, if it occurs, has no significant effect on alpha7-nAChR assembly or function.
Dunckley
T
T
Division of Neurobiology and Division of Neurology, Barrow Neurological Institute, Phoenix, Arizona 85013, USA.
Wu
J
J
Zhao
L
L
Lukas
R J
RJ
D000409:Alanine / Q000235:genetics; D002038:Bungarotoxins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D004252:DNA Mutational Analysis / Q000379:methods; D005110:Extracellular Space / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D014162:Transfection; D064569:alpha7 Nicotinic Acetylcholine Receptor
D002038:Bungarotoxins; C579167:Chrna7 protein, human; D007457:Iodine Radioisotopes; D021122:Protein Subunits; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D003545:Cysteine; D000409:Alanine