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

Abstract

The 42 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: (4)(2) and (4)(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 4 and2 subunit cDNA were transfected into human embryonic kidney 293 cells to create a novel model system of HS and LS 42 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 4 or2 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 4/2 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 42 receptors in this brain region. We report that the majority of 42 nAChRs in this brain region possess a stoichiometry of the (4)(2) LS subtype.

Structural principles of distinct assemblies of the human α4β2 nicotinic receptor

“…This conformational heterogeneity in β2 subunit structure likely further adds to the instability of the β-β interface. These structural observations suggest that the 3α:2β stoichiometry would be the predominant assembly in the absence of other factors, consistent with observations in rat motor cortex 11 and in recombinant mammalian expression 9 . In our sample preparation, we exploited nicotine and low temperature during viral transduction to boost overall receptor expression, two factors that are known to shift the stoichiometry toward the 2α:3β assembly 9 .…”

Potentiation of a neuronal nicotinic receptor via pseudo-agonist site

“…In support, W176 from the principal face of the 2 subunit contacts H142 from the complementary face the 4 subunit [49], and together the two residues could contribute to an interfacial drug binding site. In addition, NS9283 did not compete against [ 3 H]-cytisine binding to rat cortical tissue [55], a region rich in (4)3(2)2 receptors [40], suggesting it does not bind to the orthosteric sites. Nevertheless, the 2-4 subunit interfaces might not be the binding sites for NS9283, but may instead serve as transduction elements for potentiation.…”

“…Seminal studies showed that when 4 and 2 subunits were co-expressed, the agonist dose-response relationship, based on recordings of voltage clamped macroscopic current, exhibited high and low sensitivity components [12,13,15,40]. The high sensitivity component was shown to arise from receptors with two 4 and three 2 subunits, whereas the low sensitivity component was shown to arise from receptors with three 4 and two 2 subunits [13,17].…”

Interacting amino acid replacements allow poison frogs to evolve epibatidine resistance

“…The α4β2 nAChR is a pentameric protein that exhibits two different stoichiometries: a high-ACh sensitivity conformation (HS), (α4) 2 (β2) 3 , and a low-ACh sensitivity conformation (LS), (α4) 3 (β2) 2 (26, 27). To determine experimentally whether the identified AA replacements provide resistance to epibatidine, we used site-directed mutagenesis to introduce poison frog AA replacements into human nAChRs.…”