Extracellular Domain Nicotinic Acetylcholine Receptors Formed by α4 and β2 Subunits

Abstract

Models of the extracellular ligand-binding domain of nicotinic acetylcholine receptors (nAChRs), which are pentameric integral membrane proteins, are attractive for structural studies because they potentially are water-soluble and better candidates for x-ray crystallography and because their smaller size is more amenable for NMR spectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of ␣7 and muscle-type subunits. Specific design requirements leading to high structural fidelity between extracellular domain nAChRs and full-length nAChRs, however, are not well understood. To study these requirements in heteromeric nAChRs, the extracellular domains of ␣4 and ␤2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with ␣4␤2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain ␣4␤2 nAChRs formed from subunits with M1 were nearly identical to affinities of ␣4␤2 nAChRs when measured with [ 3 H]epibatidine, cytisine, nicotine, and acetylcholine. Velocity sedimentation suggested that these extracellular domain nAChRs predominantly formed pentamers. The yield of these extracellular domain nAChRs was about half the yield of ␣4␤2 nAChRs. In contrast, [3 H]epibatidine binding was not detected from the extracellular domain ␣4 and ␤2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both ␣4 and ␤2 play an important role for efficient expression of extracellular domain ␣4␤2 nAChRs that are high fidelity structural models of full-length ␣4␤2 nAChRs. Nicotinic acetylcholine receptors (nAChRs)2 are ligand-gated ion channels expressed mainly in the nervous system and at the neuromuscular junction (1-3), although they also are expressed elsewhere (4). They are members of the superfamily of nicotinoid receptors, which includes ␥-aminobutyric acid (GABA) type A and C, glycine, and serotonin 5-HT3 receptors. They contain five homologous subunits, with subunits designated ␣1-␣10, ␤1-␤4, ␦, ␥, and ⑀. Each subunit is an integral membrane protein with a large, ligand-binding N-terminal extracellular domain, four transmembrane domains (M1-M4), and a cytoplasmic loop between M3 and M4. The study of structure and dynamics of nAChRs has been motivated by their roles in normal and pathologic neurophysiology, including roles in addiction, neurodegeneration, epilepsy, myasthenia gravis, and congenital myasthenic syndromes (5).Knowledge of the structure of nAChRs has come from biochemical, electrophysiological, and imaging methods (6 -8). These methods, however, do not provide comprehensive structure at atomic resolution. A significant advance toward such information has come recently from the crystallographic structure of acetylcholine-binding protein (AChBP), a water-soluble pentamer that has been the basis for homology modeling of the extracellular...

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

“…ligand binding) complexes, as shown previously by coexpression of intact ␣4 and ␤2 subunits (41) or ECDs carrying the transmembrane M1 domain (23). Gel filtration chromatography demonstrated that the coexpressed polypeptides existed mostly as microaggregated complexes (Fig.…”

“…Coexpression of the Mutated ECDs-We then attempted to coexpress the two mutated ECDs in an effort to obtain ligandbinding pentameric ECD complexes as shown by others using coexpression of intact ␣4 and ␤2 subunits (23,34). The two mECDs were expressed with different tags (␣4-mECD-FLAG and ␤2-mECD-His 6 ) to detect the coexistence of these molecules.…”

Creating an α7 Nicotinic Acetylcholine Recognition Domain from the Acetylcholine-binding Protein

“…Using mutagenesis and affinity labeling, non-neighboring residues in the primary sequence of the protein were identified as important in ligand binding (4 -8). Several studies have shown expression of ␣7 nAChR extracellular domains (9 -11) or variants (12)(13)(14), yet none have resulted in crystal structures. Studies involving the Lymnaea stagnalis (Ls) and Aplysia californica (Ac) acetylcholine-binding proteins (AChBP) have yielded many structural analyses of the binding site for these mollusk homologues (15)(16)(17)(18)(19)(20)(21).…”

Structural answers and persistent questions about how nicotinic receptors work

“…The feasibility of extracellular domain nAChRs was supported by studies with muscle-type subunits (163)(164)(165)(166)(167), α7 subunits (168)(169)(170)(171), and α4 and β2 subunits (172). Extracellular domain muscle-type subunits without any transmembrane domains formed pentamers detected by electron microscopy (165).…”

“…Extracellular domain muscle-type subunits without any transmembrane domains formed pentamers detected by electron microscopy (165). Extracellular domain α7 and α4β2 nAChRs likely were pentamers and showed affinities for small and large ligands equal to the ligand affinities of the respective full length nAChRs (169,172). Retaining M1 on the extracellular domain, however, was required for efficient expression of extracellular domain α7 and α4β2 nAChRs.…”