The (α4)₃(β2)₂ Stoichiometry of the Nicotinic Acetylcholine Receptor Predominates in the Rat Motor Cortex

The α4β2 nicotinic acetylcholine receptor (nAChR) is important in central nervous system physiology and in mediating several of the pharmacological effects of nicotine on cognition, attention, and affective states. It is also the likely receptor that mediates nicotine addiction. This receptor assembles in two distinct stoichiometries: (α4)₂(β2)₃ and (α4)₃(β2)₂, which are referred to as high-sensitivity (HS) and low-sensitivity (LS) nAChRs, respectively, based on a difference in the potency of acetylcholine to activate them. The physiologic and pharmacological differences between these two receptor subtypes have been described in heterologous expression systems. However, the presence of each stoichiometry in native tissue currently remains unknown. In this study, different ratios of rat α4 and β2 subunit cDNA were transfected into human embryonic kidney 293 cells to create a novel model system of HS and LS α4β2 nAChRs expressed in a mammalian cell line. The HS and LS nAChRs were characterized through pharmacological and biochemical methods. Isolation of surface proteins revealed higher amounts of α4 or β2 subunits in the LS or HS nAChR populations, respectively. In addition, sazetidine-A displayed different efficacies in activating these two receptor stoichiometries. Using this model system, a neurophysiological "two-concentration" acetylcholine or carbachol paradigm was developed and validated to determine α4/β2 subunit stoichiometry. This paradigm was then used in layers I-IV of slices of the rat motor cortex to determine the percent contribution of HS and LS α4β2 receptors in this brain region. We report that the majority of α4β2 nAChRs in this brain region possess a stoichiometry of the (α4)₃(β2)₂ LS subtype.