Soluble, Oligomeric, and Ligand-binding Extracellular Domain of the Human α7 Acetylcholine Receptor Expressed in Yeast

Abstract

The N-terminal extracellular domain (ECD; amino acids 1-208) of the neuronal nicotinic acetylcholine receptor (AChR) ␣7 subunit, the only human AChR subunit known to assemble as a homopentamer, was expressed as a glycosylated form in the yeast Pichia pastoris in order to obtain a native-like model of the extracellular part of an intact pentameric nicotinic AChR. This molecule, ␣7-ECD, although able to bind the specific ligand ␣-bungarotoxin, existed mainly in the form of microaggregates. Substitution of Cys-116 in the ␣7-ECD with serine led to a decrease in microaggregate size. A second mutant form, ␣7-ECD(C116S,Cys-loop), was generated in which, in addition to the C116S mutation, the hydropho- ؊7 M, K i ‫؍‬ 1 ؋ 10 ؊5 M, and K i ‫؍‬ 0.9 ؋ 10 ؊2 M, respectively). All three constructs were expressed as glycosylated forms, but in vitro deglycosylation reduced the heterogeneity without affecting their ligand binding properties. These results show that ␣7-ECD(C116S,Cys-loop) was expressed in P. pastoris as an oligomer (probably a pentamer) with a near native conformation and that its deglycosylated form seems to be suitable starting material for structural studies on the ligand-binding domain of a neurotransmitter receptor.

Extracellular Domain Nicotinic Acetylcholine Receptors Formed by α4 and β2 Subunits

“…M1, however, played an important role for expressing these extracellular domain nAChRs. These results, with the properties of extracellular domain ␣7 nAChRs (33,34) and evidence of oligomerization of extracellular domains from ␣1 and ␦ subunits (31,32), from extracellular domains of muscle-type subunits (35), from chimeric ␣7/AChBP subunits (26), from glycine receptor subunits (68,69), and from GABA A receptor subunits (70), suggest that producing extracellular domain receptors might be possible for many subunits throughout the nicotinoid family. Expression of extracellular domain nicotinoid receptors with high structural fidelity, however, might be more feasible with M1 included in the subunit design than without M1.…”

“…Third, chimeric extracellular domain nAChR subunits that include sequences from AChBP might lead to water-soluble pentamers with high structural fidelity to the parent full-length nAChRs. Studies of chimeric, water-soluble extracellular domains from ␣7 and AChBP sequences (26) and chimeric ion channels containing a chimeric AChBP/5-hydroxytryptamine (serotonin) receptor subunit type 3A extracellular domain and the pore domain from the 5-hydroxytryptamine (serotonin) receptor subunit type 3A subunit (79) have begun to explore the feasibility of this approach. Fourth, increasing host factors that contribute to subunit folding and assembly also might improve the efficiency of producing water-soluble nAChRs.…”

“…In that case, shorter designs of ␣4WS and ␤2WS might be better suited for expressing water-soluble extracellular domain ␣4␤2 nAChRs. Increasing the hydrophilic character of the Cys loop in the extracellular domain is another potentially favorable change in the designs of ␣4WS and ␤2WS, based on the increased water solubility of a chimeric ␣7 extracellular domain with a more hydrophilic Cys loop sequence taken from AChBP (26).…”

“…Their yield, however, was substantially smaller than the yield of detergent-solubilized, extracellular domain ␣7 nAChRs that contained M1, suggesting that M1 was important for expressing these extracellular domain nAChRs. Milligram amounts of a water-soluble, extracellular domain chimera containing ␣7 and AChBP sequences were produced in yeast (26). These extracellular domains formed oligomers that likely were pentamers.…”

Expression of Water-soluble, Ligand-binding Concatameric Extracellular Domains of the Human Neuronal Nicotinic Receptor α4 and β2 Subunits in the Yeast Pichia pastoris

“…In fact, to our knowledge, no other ECD of any AChR with a high affinity for small ligands has been constructed, with the exception of the protein ligand ␣-bungarotoxin, the binding of which does not involve a subunit interface and thus binds with high affinity to ␣1 and ␣7 ECDs (28,29).…”