Probing the allosteric role of the alpha 5 subunit of alpha 3 beta 4 alpha 5 nicotinic acetylcholine receptors by functionally selective modulators and ligands

Abstract

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 alpha 3 beta 4 alpha 5-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 alpha 3 beta 4 alpha 5 signaling. In this study, we describe the development of homogeneous HEK-293 cell clones of alpha 3 beta 4 and alpha 3 beta 4 alpha 5 receptors appropriate for drug screening and characterizing biochemical and pharmacological properties of incorporated alpha 5 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 alpha 3 subunits in approximately 98% of plasma membrane receptor pentamers, indicating a 2/3 subunit expression ratio of alpha 3 to beta 4 alone or to coexpressed beta 4 and alpha 5. With prolonged nicotine exposure, the plasma membrane expression of receptors with and without incorporated alpha 5 increased. Whereas alpha 5 subunit expression decreased the cell calcium response to nicotine and reduced plasma membrane receptor number, it partially protected receptors from nicotine mediated desensitization. Hit compounds from both libraries suggest the alpha 5 and alpha 5-D398N subunits allosterically modify the behavior of nicotine at the parent alpha 3 beta 4 nicotinic acetylcholine receptor. These studies identify pharmacological tools from two distinct classes of drugs, antagonists and modifiers that are alpha 5 and alpha 5-D398N subtype selective that provide a means to characterize the role of the CHRNAS/A3/B4 gene cluster in smoking and cancer.