Probing the Allosteric Role of the α5 Subunit of α3β4α5 Nicotinic Acetylcholine Receptors by Functionally Selective Modulators and Ligands

Nicotinic acetylcholine receptors regulate the nicotine dependence encountered with cigarette smoking, and this has stimulated a search for drugs binding the responsible receptor subtypes. Studies link a gene cluster encoding for α3β4α5-D398N nicotinic acetylcholine receptors to lung cancer risk as well as link a second mutation in this cluster to an increased risk for nicotine dependence. However, there are currently no recognized drugs for discriminating α3β4α5 signaling. In this study, we describe the development of homogeneous HEK-293 cell clones of α3β4 and α3β4α5 receptors appropriate for drug screening and characterizing biochemical and pharmacological properties of incorporated α5 subunits. Clones were assessed for plasma membrane expression of the individual receptor subunits by mass spectrometry and immunochemistry, and their calcium flux was measured in the presence of a library of kinase inhibitors and a focused library of acetylcholine receptor ligands. We demonstrated an incorporation of two...