Mutational analysis of roles for extracellular cysteine residues in the assembly and function of human α7-nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChR) containing α7 subunits self-assemble into simple, homopentameric complexes. However, successful heterologous expression of functional α7-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-HT₃ receptor, and nAChR subunits contain a pair of highly conserved cysteine residues (C150 and C164 for α7 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 α7 (and α8) 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 α7-nAChR. Results indicate that mutation of C138, but not of C150 or C164, yields an nAChR that can assemble to form ¹²⁵I-labeled α-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 α7-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 α7-nAChR. These results suggest that disulfide bond formation between C138 and either C150 or C164, if it occurs, has no significant effect on α7-nAChR assembly or function.