Affinity purification of a chimeric nicotinic acetylcholine receptor in the agonist and antagonist bound states.

Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels that mediate fast signal transmission at synapses. These receptors are members of a large family of pentameric ion channels that are of active medical interest. An expression system utilizing a chimerical construct of the N-terminal extracellular ligand binding domain of alpha7 type nAChR and the C-terminal transmembrane portion of 5HT3 type receptor resulted high level of expressions. Two ligand affinity chromatography purification methods for this receptor have been developed. One method relies on the covalent immobilization of a high affinity small molecule alpha7 nAChR agonist, (R)-5-(4-aminophenyl)-N-(quinuclidin-3-yl) furan-2-carboxamide, and the other uses mono biotinylated alpha-bungarotoxin, an antagonist, that forms a quasi-irreversible complex with alpha7 nAChR. Detergent solubilized alpha7/5HT(3) chimeric receptors were selectively retained on the affinity resins and could be eluted with free ligand or biotin. The proteins purified by both methods were characterized by gel electrophoresis, mass spectra, amino acid composition analysis, and N-terminal sequence determination. These analyses confirmed the isolation of a mature alpha7/5HT(3) receptor with the signal peptide removed. These results suggest a scalable path forward to generate multi-milligram amounts of purified complexes for additional studies including protein crystallization.

Discovery of a protein sequence in the N-terminal region of the human neuronal alpha7 nicotinic acetylcholine receptor involved in homomeric interactions.

The neuronal nicotinic acetylcholine receptor subunit, alpha7, can form homopentameric receptor/ion channel complexes. Potential contributions of its N-terminal region to homomeric interactions were investigated, in comparison with the corresponding region of an analogous heteromeric subunit, alpha3. Recombinant chimeras were prepared upon engineering the N-terminal alpha7 (M1-V224) or alpha3 (M1-S232) sequence into the background of another homomeric mouse 5-hydroxytryptamine3 (5-HT)(3) receptor. The alpha7/5-HT(3) chimera, when expressed heterologously in a human epithelial cell line, SH-EP1, robustly expressed alpha-bungarotoxin binding sites as homooligomers while the alpha3/5-HT(3) did not produce epibatidine (non-selective ligand) binding sites, and did not interfere the alpha7/5-HT3 phenotype, upon co-expression. Yeast two hybrid assays with the N-terminal regions showed positive responses between alpha7:alpha7, but not between alpha7:alpha3 and alpha3:alpha3. Similar assays with the alpha7 N-terminal region and its five smaller fragments (G23-N46, D47-N90, V91-N133, S134-M182and Q183-V224) revealed that the G23-N46 sequence is involved in homomeric interactions. Replacement of the corresponding region of the alpha3/5-HT(3) chimera with the alpha7 G23-N46 sequence conferred a dominant negative role on the chimera, by abolishing the alpha7/5-HT(3) phenotype. These results support the view that the G23-N46 portion of the alpha7 N-terminal region may contribute to receptor homooligomerizations.