Extracellular Domain Nicotinic Acetylcholine Receptors Formed by α4 and β2 Subunits

Alexandra M Person,Kathy L Bills,Hong Liu,Shaleen K Botting,Jon Lindstrom,Gregg B Wells

doi:10.1074/jbc.m505087200

Alexandra M Person, Kathy L Bills + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m505087200

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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 alpha7 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 alpha4 and beta2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with alpha4beta2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain alpha4beta2 nAChRs formed from subunits with M1 were nearly identical to affinities of alpha4beta2 nAChRs when measured with [3H]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 alpha4beta2 nAChRs. In contrast, [3H]epibatidine binding was not detected from the extracellular domain alpha4 and beta2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both alpha4 and beta2 play an important role for efficient expression of extracellular domain alpha4beta2 nAChRs that are high fidelity structural models of full-length alpha4beta2 nAChRs.

Highlights

Nicotinic acetylcholine receptors2 are ligand-gated ion channels expressed mainly in the nervous system and at the neuromuscular junction [1,2,3], they are expressed elsewhere [4]
The nearly identical ligand affinities of ␣4M1/␤2M1 Nicotinic acetylcholine receptors (nAChRs), the predominant species for ␣4␤2 nAChRs, and the assembly of ␣4M1 and ␤2M1 subunits into multimers that likely are pentamers suggest that extracellular domains with the M1 domains of ␣4 and ␤2 subunits form high fidelity structural models of ␣4␤2 nAChRs
The values of dissociation constants for binding between ␣4␤2 nAChRs and [3H]epibatidine, cytisine, nicotine, and acetylcholine are similar to values reported from other laboratories (48 –51, 65– 67)

Summary

EXPERIMENTAL PROCEDURES

Design of Subunit DNA Plasmids—The cDNAs for human ␣4 subunit [37] and human ␤2 subunit [38] were cloned into pSP64 poly(A) (Promega). The microwells were incubated overnight at 4 °C to allow binding of epitope-tagged subunits and washed with ice-cold buffer B. Values for Kdco and Kdco were estimated by least squares fitting of the equation RE ϩ 2 ϫ RE2 (total concentration of bound [3H]epibatidine) simultaneously to all data sets of [3H]epibatidine binding, where RE and RE2 are defined by Equations 3 and 4. Values for Kdin and Kdin were estimated by least squares fitting of the equation R [1]E ϩ R [2]E (total concentration of bound [3H]epibatidine) from Equations 6 and 7 simultaneously to all relevant data sets. Equation 9 describes the concentration of the [3H]epibatidine-binding species for the one-site competitive inhibition model, Bound3Hepibatidine ϭ Rt/͑1 ϩIt/IC50͒nϩ C (Eq 12). Relative yield helped control for variations in protein expression that depended on time of year or the particular frog from which oocytes were harvested

RESULTS

Two independent sitese

TABLE TWO

TABLE THREE

DISCUSSION