Detection of α7 nicotinic acetylcholine receptors with the aid of antibodies and toxins

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) containing α7 subunit are well represented in the brain and some non-neuronal tissues, and their malfunctioning is associated with diverse pathologies. Therefore, detection and quantification of α7 nAChR are important tasks. The affinity-purified antibodies were prepared against the 1–23 and 179–190 fragments of the human and rat α7 nAChR extracellular domain. The specificity and selectivity of these α7 (1–23) and α7 (179–190) antibodies was tested by ELISA in model systems: the E. coli-expressed α7 subunit extracellular domain and the pituitary cell line GH4C1 stably expressing human α7 nAChR. On the rat brain slices two antibodies and biotinylated α-cobratoxin specifically stained the hippocampus region known to be rich in α7 nAChR. Western blot analysis revealed that in the human thalamus membranes and in rat brain membranes, antibodies α7 (1–23) stained a single band of 62 kDa, while the α7 (179–190) antibodies stained a doublet of 53–54 kDa. The results obtained show that utilization of model systems and a combination of several antibodies with appropriately labeled toxins may provide better ways for detection of α7 nAChR.