The neuronal alpha 7 nicotinic acetylcholine receptor is a homo-pentameric ligand-gated ion channel that is a promising drug target for cognitive deficits in Alzheimer's disease and schizophrenia. We have previously described 11C-NS14492 as a suitable agonist radioligand for in vivo positron emission tomography (PET) occupancy studies of the alpha 7 nicotinic receptor in the pig brain. In order to investigate the utility of the same compound for in vitro studies, 3H-NS14492 was synthesized and its binding properties were characterized using in vitro autoradiography and homogenate binding assays in pig frontal cortex. 3H-NS14492 showed specific binding to alpha 7 nicotinic receptors in autoradiography, revealing a dissociation constant (Kd) of 2.1 ± 0.7 nM and a maximum number of binding sites (Bmax) of 15.7±2.0 fmol/mg tissue equivalent. Binding distribution was similar to that of another selective ligand 125I-alpha-bungarotoxin (125I-BTX) in autoradiography, and unlabeled NS14492 displaced 125I-BTX with an inhibition constant (Ki) of 23 nM. 3H-NS14492 bound to alpha 7 nicotinic receptors in homogenized pig frontal cortex with a Kd of 0.8±0.3 nM and a Bmax of 30.2±11.6 fmol/mg protein. This binding assay further revealed the Ki rank order for a number of alpha 7 nicotinic receptor agonists, and positive allosteric modulators (PAMs). Further, we saw increased binding of 3H-NS14492 to pig frontal cortex membranes when co-incubated with PNU-120596, a type II PAM. Taken together, these findings show that 3H-NS14492 is a useful new in vitro radioligand for the pig alpha 7 nicotinic receptor. © 2015 Elsevier B.V. All rights reserved.
- Binding assay
- PET ligand